Method of detecting and/or identifying adeno-associated virus (aav) sequences and isolating novel sequences identified thereby

ABSTRACT

Adeno-associated virus rh.20 sequences, vectors containing same, and methods of use are provided.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icosahedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus,Dependovirus, because the virus was discovered as a contaminant inpurified adenovirus stocks. AAV's life cycle includes a latent phase atwhich AAV genomes, after infection, are site specifically integratedinto host chromosomes and an infectious phase in which, following eitheradenovirus or herpes simplex virus infection, the integrated genomes aresubsequently rescued, replicated, and packaged into infectious viruses.The properties of non-pathogenicity, broad host range of infectivity,including non-dividing cells, and potential site-specific chromosomalintegration make AAV an attractive tool for gene transfer.

Recent studies suggest that AAV vectors may be the preferred vehicle forgene therapy. To date, there have been 6 different serotypes of AAVsisolated from human or non-human primates (NHP) and well characterized.Among them, human serotype 2 is the first AAV that was developed as agene transfer vector; it has been widely used for efficient genetransfer experiments in different target tissues and animal models.Clinical trials of the experimental application of AAV2 based vectors tosome human disease models are in progress, and include such diseases ascystic fibrosis and hemophilia B.

What are desirable are AAV-based constructs for gene delivery.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a novel method of detecting andidentifying AAV sequences from cellular DNAs of various human andnon-human primate (NHP) tissues using bioinformatics analysis, PCR basedgene amplification and cloning technology, based on the nature oflatency and integration of AAVs in the absence of helper virusco-infection.

In another aspect, the invention provides method of isolating novel AAVsequences detected using the above described method of the invention.The invention further comprises methods of generating vectors based uponthese novel AAV serotypes, for serology and gene transfer studies solelybased on availability of capsid gene sequences and structure of rep/capgene junctions.

In still another aspect, the invention provides a novel method forperforming studies of serology, epidemiology, biodistribution and modeof transmission, using reagents according to the invention, whichinclude generic sets of primers/probes and quantitative real time PCR.

In yet another aspect, the invention provides a method of isolatingcomplete and infectious genomes of novel AAV serotypes from cellular DNAof different origins using RACE and other molecular techniques.

In a further aspect, the invention provides a method of rescuing novelserotypes of AAV genomes from human and NHP cell lines using adenovirushelpers of different origins.

In still a further aspect, the invention provides novel AAV serotypes,vectors containing same, and methods of using same.

These and other aspects of the invention will be readily apparent fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1AAAR provide an alignment of the nucleic acidsequences encoding at least the cap proteins for the AAV serotypes. Thefull-length sequences including the ITRs, the rep region, and the capsidregion are provided for novel AAV serotype 7 [SEQ ID NO:11], and forpreviously published AAV1 [SEQ IN NO:6], AAV2 [SEQ ID NO:7]; and AAV3[SEQ ID NO:8]. Novel AAV serotypes AAV8 [SEQ ID NO:4] and AAV9 [SEQ IDNO:5] are the subject of co-filed applications. The other novel clonesof the invention provided in this alignment include: 42-2 [SEQ ID NO:9],42-8 [SEQ ID NO:27], 42-15 [SEQ ID NO:28], 42-5b [SEQ ID NO: 29], 42-1b[SEQ ID NO:30]; 42-13 [SEQ ID NO: 31], 42-3a [SEQ ID NO: 32], 42-4 [SEQID NO:33], 42-5a [SEQ ID NO: 34], 42-10 [SEQ ID NO:35], 42-3b [SEQ IDNO: 36], 42-11 [SEQ ID NO: 37], 42-6b [SEQ ID NO:38], 43-1 [SEQ ID NO:39], 43-5 [SEQ ID NO: 40], 43-12 [SEQ ID NO:41], 43-20 [SEQ ID NO:42],43-21 [SEQ ID NO: 43], 43-23 [SEQ ID NO:44], 43-25 [SEQ ID NO: 45], 44,1[SEQ ID NO:47], 44.5 [SEQ ID NO:47], 223.10 [SEQ ID NO:48], 223.2 [SEQID NO:49], 223.4 [SEQ ID NO:50], 223.5 [SEQ ID NO: 51], 223.6 [SEQ IDNO: 52], 223.7 [SEQ ID NO: 53], A3.4 [SEQ ID NO: 54], A3.5 [SEQ IDNO:55], A3.7 [SEQ ID NO: 56], A3.3 [SEQ ID NO:57], 42.12 [SEQ ID NO:58], 44.2 [SEQ ID NO: 59]. The nucleotide sequences of the signatureregions of AAV10 [SEQ ID NO: 117], AAV11 [SEQ ID NO: 118] and AAV12 [SEQID NO:119] are provided in this figure. Critical landmarks in thestructures of AAV genomes are shown. Gaps are demonstrated by dots. The3′ ITR of AAV1 [SEQ ID NO:6] is shown in the same configuration as inthe published sequences. TRS represents terminal resolution site. Noticethat AAV7 is the only AAV reported that uses GTG as the initiation codonfor VP3.

FIGS. 2A through 2M are an alignment of the amino acid sequences of theproteins of the vp1 capsid proteins of previously published AAVserotypes 1 [SEQ ID NO:64], AAV2 [SEQ ID NO:70], AAV3 [SEQ ID NO: 71],AAV4 [SEQ ID NO:63], AAV 5 [SEQ ID NO:114], and AAV6 [SEQ ID NO:65] andnovel AAV sequences of the invention, including: C1 [SEQ ID NO:60], C2[SEQ ID NO:61], C5 [SEQ ID NO:62], A3-3 [SEQ ID NO:66], A3-7 [SEQ IDNO:67], A3-4 [SEQ ID NO:68], A3-5 [SEQ ID NO: 69], 3.3b [SEQ ID NO: 62],223.4 [SEQ ID NO: 73], 223-5 [SEQ ID NO:74], 223-10 [SEQ ID NO:75],223-2 [SEQ ID NO:76], 223-7 [SEQ ID NO: 77], 223-6 [SEQ ID NO: 78], 44-1[SEQ ID NO: 79], 44-5 [SEQ ID NO:80], 44-2 [SEQ ID NO:81], 42-15 [SEQ IDNO: 84], 42-8 [SEQ ID NO: 85], 42-13 [SEQ ID NO:86], 42-3A [SEQ IDNO:87], 42-4 [SEQ ID NO:88], 42-5A [SEQ ID NO:89], 42-1B [SEQ ID NO:90],42-5B [SEQ ID NO:91], 43-1 [SEQ ID NO: 92], 43-12 [SEQ ID NO: 93], 43-5[SEQ ID NO:94], 43-21 [SEQ ID NO:96], 43-25 [SEQ ID NO: 97], 43-20 [SEQID NO:99], 24.1 [SEQ ID NO: 101], 42.2 [SEQ ID NO:102], 7.2 [SEQ ID NO:103], 27.3 [SEQ ID NO: 104], 16.3 [SEQ ID NO: 105], 42.10 [SEQ ID NO:106], 42-3B [SEQ ID NO: 107], 42-11 [SEQ ID NO: 108], F1 [SEQ ID NO:109], F5 [SEQ ID NO: 110], F3 [SEQ ID NO:111], 42-6B [SEQ ID NO: 112],42-12 [SEQ ID NO: 113]. Novel serotypes AAV8 [SEQ ID NO:95] and AAV9[SEQ ID NO:100] are the subject of co-filed patent applications.

FIGS. 3A through 3C provide the amino acid sequences of the AAV7 repproteins [SEQ ID NO:3].

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the inventors have found a method which takesadvantage of the ability of adeno-associated virus (AAV) to penetratethe nucleus, and, in the absence of a helper virus co-infection, tointegrate into cellular DNA and establish a latent infection. Thismethod utilizes a polymerase chain reaction (PCR)-based strategy fordetection, identification and/or isolation of sequences of AAVs fromDNAs from tissues of human and non-human primate origin as well as fromother sources. Advantageously, this method is also suitable fordetection, identification and/or isolation of other integrated viral andnon-viral sequences, as described below.

The invention further provides nucleic acid sequences identifiedaccording to the methods of the invention. One such adeno-associatedvirus is of a novel serotype, termed herein serotype 7 (AAV7). Othernovel adeno-associated virus serotypes provided herein include AAV10,AAV11, and AAV12. Still other novel AAV serotypes identified accordingto the methods of the invention are provided in the presentspecification. See, Figures and Sequence Listing, which is incorporatedby reference.

Also provided are fragments of these AAV sequences. Among particularlydesirable AAV fragments are the cap proteins, including the vp1, vp2,vp3, the hypervariable regions, the rep proteins, including rep 78, rep68, rep 52, and rep 40, and the sequences encoding these proteins. Eachof these fragments may be readily utilized in a variety of vectorsystems and host cells. Such fragments may be used alone, in combinationwith other AAV sequences or fragments, or in combination with elementsfrom other AAV or non-AAV viral sequences. In one particularly desirableembodiment, a vector contains the AAV cap and/or rep sequences of theinvention.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as AClustal W≅, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similar programs are available for amino acidsequences, e.g., the “Clustal X” program. Generally, any of theseprograms are used at default settings, although one of skill in the artcan alter these settings as needed. Alternatively, one of skill in theart can utilize another algorithm or computer program which provides atleast the level of identity or alignment as that provided by thereferenced algorithms and programs.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or anopen reading frame thereof, or another suitable fragment which is atleast 15 nucleotides in length. Examples of suitable fragments aredescribed herein.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid, there is amino acid sequence identity in atleast about 95 to 99% of the aligned sequences. Preferably, the homologyis over full-length sequence, or a protein thereof, e.g., a cap protein,a rep protein, or a fragment thereof which is at least 8 amino acids, ormore desirably, at least 15 amino acids in length. Examples of suitablefragments are described herein.

By the term “highly conserved” is meant at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. Identity is readily determined by one of skill in the art byresort to algorithms and computer programs known by those of skill inthe art.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome,the full-length of a gene coding sequence, or a fragment of at leastabout 500 to 5000 nucleotides, is desired. However, identity amongsmaller fragments, e.g. of at least about nine nucleotides, usually atleast about 20 to 24 nucleotides, at least about 28 to 32 nucleotides,at least about 36 or more nucleotides, may also be desired. Similarly,“percent sequence identity” may be readily determined for amino acidsequences, over the full-length of a protein, or a fragment thereof.Suitably, a fragment is at least about 8 amino acids in length, and maybe up to about 700 amino acids. Examples of suitable fragments aredescribed herein.

The AAV sequences and fragments thereof are useful in production ofrAAV, and are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. The invention further provides nucleic acidmolecules, gene delivery vectors, and host cells which contain the AAVsequences of the invention.

As described herein, the vectors of the invention containing the AAVcapsid proteins of the invention are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other AAV serotype based vectors, as well as otherviral vectors. The rAAV vectors of the invention are particularlyadvantageous in rAAV readministration and repeat gene therapy.

These and other embodiments and advantages of the invention aredescribed in more detail below. As used throughout this specificationand the claims, the terms Acomprising≅ and “including” and theirvariants are inclusive of other components, elements, integers, stepsand the like. Conversely, the term “consisting” and its variants isexclusive of other components, elements, integers, steps and the like.

I. Methods of the Invention

A. Detection of Sequences Via Molecular Cloning

In one aspect, the invention provides a method of detecting and/oridentifying target nucleic acid sequences in a sample. This method isparticularly well suited for detection of viral sequences which areintegrated into the chromosome of a cell, e.g., adeno-associated viruses(AAV) and retroviruses, among others. The specification makes referenceto AAV, which is exemplified herein. However, based on this information,one of skill in the art may readily perform the methods of the inventionon retroviruses [e.g., feline leukemia virus (FeLV), HTLVI and HTLVII],and lentivirinae [e.g., human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), feline immunodeficiency virus (FIV),equine infectious anemia virus, and spumavirinal)], among others.Further, the method of the invention may also be used for detection ofother viral and non-viral sequences, whether integrated ornon-integrated into the genome of the host cell.

As used herein, a sample is any source containing nucleic acids, e.g.,tissue, tissue culture, cells, cell culture, and biological fluidsincluding, without limitation, urine and blood. These nucleic acidsequences may be DNA or RNA from plasmids, natural DNA or RNA from anysource, including bacteria, yeast, viruses, and higher organisms such asplants or animals. DNA or RNA is extracted from the sample by a varietyof techniques known to those of skill in the art, such as thosedescribed by Sambrook, Molecular Cloning: A Laboratory Manual (New York:Cold Spring Harbor Laboratory). The origin of the sample and the methodby which the nucleic acids are obtained for application of the method ofthe invention is not a limitation of the present invention. Optionally,the method of the invention can be performed directly on the source ofDNA, or on nucleic acids obtained (e.g., extracted) from a source.

The method of the invention involves subjecting a sample containing DNAto amplification via polymerase chain reaction (PCR) using a first setof primers specific for a first region of double-stranded nucleic acidsequences, thereby obtaining amplified sequences.

As used herein, each of the Aregions≅ is predetermined based upon thealignment of the nucleic acid sequences of at least two serotypes (e.g.,AAV) or strains (e.g., lentiviruses), and wherein each of said regionsis composed of sequences having a 5′ end which is highly conserved, amiddle which is preferably, but necessarily, variable, and a 3′ endwhich is highly conserved, each of these being conserved or variablerelative to the sequences of the at least two aligned AAV serotypes.Preferably, the 5′ and/or 3′ end is highly conserved over at least about9, and more preferably, at least 18 base pairs (bp). However, one orboth of the sequences at the 5= or 3=end may be conserved over more than18 bp, more than 25 bp, more than 30 bp, or more than 50 bp at the 5′end. With respect to the variable region, there is no requirement forconserved sequences, these sequences may be relatively conserved, or mayhave less than 90, 80, or 70% identity among the aligned serotypes orstrains.

Each of the regions may span about 100 bp to about 10 kilobase pairs inlength. However, it is particularly desirable that one of the regions isa Asignature region≅, i.e., a region which is sufficiently unique topositively identify the amplified sequence as being from the targetsource. For example, in one embodiment, the first region is about 250 bpin length, and is sufficiently unique among known AAV sequences, that itpositively identifies the amplified region as being of AAV origin.Further, the variable sequences within this region are sufficientlyunique that can be used to identify the serotype from which theamplified sequences originate. Once amplified (and thereby detected),the sequences can be identified by performing conventional restrictiondigestion and comparison to restriction digestion patterns for thisregion in any of AAV1, AAV2, AAV3, AAV4, AAV5, or AAV6, or that of AAV7,AAV10, AAV11, AAV12, or any of the other novel serotypes identified bythe invention, which is predetermined and provided by the presentinvention.

Given the guidance provided herein, one of skill in the art can readilyidentify such regions among other integrated viruses to permit readydetection and identification of these sequences. Thereafter, an optimalset of generic primers located within the highly conserved ends can bedesigned and tested for efficient amplification of the selected regionfrom samples. This aspect of the invention is readily adapted to adiagnostic kit for detecting the presence of the target sequence (e.g.,AAV) and for identifying the AAV serotype, using standards which includethe restriction patterns for the AAV serotypes described herein orisolated using the techniques described herein. For example, quickidentification or molecular serotyping of PCR products can beaccomplished by digesting the PCR products and comparing restrictionpatterns.

Thus, in one embodiment, the “signature region” for AAV spans about bp2800 to about 3200 of AAV 1 [SEQ ID NO:6], and corresponding base pairsin AAV 2, AAV3, AAV4, AAV5, and AAV6. More desirably, the region isabout 250 bp, located within bp 2886 to about 3143 bp of AAV 1 [SEQ IDNO:6], and corresponding base pairs in AAV 2 [SEQ ID NO:7], AAV3 [SEQ IDNO8], and other AAV serotypes. See, FIG. 1. To permit rapid detection ofAAV in the sample, primers which specifically amplify this signatureregion are utilized. However, the present invention is not limited tothe exact sequences identified herein for the AAV signature region, asone of skill in the art may readily alter this region to encompass ashorter fragment, or a larger fragment of this signature region.

The PCR primers are generated using techniques known to those of skillin the art. Each of the PCR primer sets is composed of a 5′ primer and a3′ primer. See, e.g., Sambrook et al, cited herein. The term “primer”refers to an oligonucleotide which acts as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced. The primer is preferably single stranded. However, if a doublestranded primer is utilized, it is treated to separate its strandsbefore being used to prepare extension products. The primers may beabout 15 to 25 or more nucleotides, and preferably at least 18nucleotides. However, for certain applications shorter nucleotides,e.g., 7 to 15 nucleotides are utilized.

The primers are selected to be sufficiently complementary to thedifferent strands of each specific sequence to be amplified to hybridizewith their respective strands. Therefore, the primer sequence need notreflect the exact sequence of the region being amplified. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence being completelycomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to be amplified to hybridize therewith and form a template forsynthesis of the extension product of the other primer.

The PCR primers for the signature region according to the invention arebased upon the highly conserved sequences of two or more alignedsequences (e.g., two or more AAV serotypes). The primers can accommodateless than exact identity among the two or more aligned AAV serotypes atthe 5′ end or in the middle. However, the sequences at the 3′ end of theprimers correspond to a region of two or more aligned AAV serotypes inwhich there is exact identity over at least five, preferably, over atleast nine base pairs, and more preferably, over at least 18 base pairsat the 3′ end of the primers. Thus, the 3′ end of the primers iscomposed of sequences with 100% identity to the aligned sequences overat least five nucleotides. However, one can optionally utilize one, two,or more degenerate nucleotides at the 3′ end of the primer.

For example, the primer set for the signature region of AAV was designedbased upon a unique region within the AAV capsid, as follows. The 5′primer was based upon nt 2867-2891 of AAV2 [SEQ ID NO:7],5′-GGTAATTCCTCCGGAAATTGGCATT3′. See, FIG. 1. The 3′ primer was designedbased upon nt 3096-3122 of AAV2 [SEQ ID NO:7],5′-GACTCATCAACAACAACTGGGGATTC-3′. However, one of skill in the art mayhave readily designed the primer set based upon the correspondingregions of AAV 1, AAV3, AAV4, AAV5, AAV6, or based upon the informationprovided herein, AAV7, AAV10, AAV11, AAV12, or another novel AAV of theinvention. In addition, still other primer sets can be readily designedto amplify this signature region, using techniques known to those ofskill in the art.

B. Isolation of Target Sequences

As described herein, the present invention provides a first primer setwhich specifically amplifies the signature region of the targetsequence, e.g., an AAV serotype, in order to permit detection of thetarget. In a situation in which further sequences are desired, e.g., ifa novel AAV serotype is identified, the signature region may beextended. Thus, the invention may further utilize one or more additionalprimer sets.

Suitably, these primer sets are designed to include either the 5′ or 3′primer of the first primer set and a second primer unique to the primerset, such that the primer set amplifies a region 5′ or 3′ to thesignature region which anneals to either the 5′ end or the 3′ end of thesignature region. For example, a first primer set is composed of a 5′primer, P1 and a 3′ primer P2 to amplify the signature region. In orderto extend the signature region on its 3′ end, a second primer set iscomposed of primer P1 and a 3′ primer P4, which amplifies the signatureregion and contiguous sequences downstream of the signature region. Inorder to extend the signature region on its 5′ end, a third primer setis composed of a 5′ primer, P5, and primer P2, such that the signatureregion and contiguous sequences upstream of the signature region areamplified. These extension steps are repeated (or performed at the sametime), as needed or desired. Thereafter, the products results from theseamplification steps are fused using conventional steps to produce anisolated sequence of the desired length.

The second and third primer sets are designed, as with the primer setfor the signature region, to amplify a region having highly conservedsequences among the aligned sequences. Reference herein to the term“second” or “third” primer set is for each of discussion only, andwithout regard to the order in which these primers are added to thereaction mixture, or used for amplification. The region amplified by thesecond primer set is selected so that upon amplification it anneals atits 5′ end to the 3′ end of the signature region. Similarly, the regionamplified by the third primer set is selected so that upon amplificationit anneals at its 3′ end anneals to the 5′ end of the signature region.Additional primer sets can be designed such that the regions which theyamplify anneal to the either the 5′ end or the 3′ end of the extensionproducts formed by the second or third primer sets, or by subsequentprimer sets.

For example, where AAV is the target sequence, a first set of primers(P1 and P2) are used to amplify the signature region from the sample. Inone desirable embodiment, this signature region is located within theAAV capsid. A second set of primers (P1 and P4) is used to extend the 3′end of the signature region to a location in the AAV sequence which isjust before the AAV 3′ ITR, i.e., providing an extension productcontaining the entire 3′ end of the AAV capsid when using the signatureregion as an anchor. In one embodiment, the P4 primer corresponds to nt4435 to 4462 of AAV2 [SEQ ID NO:7], and corresponding sequences in theother AAV serotypes. This results in amplification of a region of about1.6 kb, which contains the 0.25 kb signature region. A third set ofprimers (P3 and P2) is used to extend the 5′ end of signature region toa location in the AAV sequences which is in the 3′ end of the rep genes,i.e., providing an extension product containing the entire 5′ end of theAAV capsid when using the signature region as an anchor. In oneembodiment, the P3 primer corresponds to nt 1384 to 1409 of AAV2 [SEQ IDNO:7], and corresponding sequences in the other AAV serotypes. Thisresults in amplification of a region of about 1.7 kb, which contains the0.25 kb signature region. Optionally, a fourth set of primers are usedto further extend the extension product containing the entire 5′ end ofthe AAV capsid to also include the rep sequences. In one embodiment, theprimer designated P5 corresponds to nt 108 to 133 of AAV2 [SEQ ID NO:7],and corresponding sequences in the other AAV serotypes and is used inconjunction with the P2 primer.

Following completion of the desired number of extension steps, thevarious extension products are fused, making use of the signature regionas an anchor or marker, to construct an intact sequence. In the exampleprovided herein, AAV sequences containing, at a minimum, an intact AAVcap gene are obtained. Larger sequences may be obtained, depending uponthe number of extension steps performed.

Suitably, the extension products are assembled into an intact AAVsequence using methods known to those of skill in the art. For example,the extension products may be digested with DraIII, which cleaves at theDraIII site located within the signature region, to provide restrictionfragments which are re-ligated to provide products containing (at aminimum) an intact AAV cap gene. However, other suitable techniques forassembling the extension products into an intact sequence may beutilized. See, generally, Sambrook et al, cited herein.

As an alternative to the multiple extension steps described above,another embodiment of the invention provides for direct amplification ofa 3.1 kb fragment which allows isolation of full-length cap sequences.To directly amplify a 3.1 kb full-length cap fragment from NHP tissueand blood DNAs, two other highly conserved regions were identified inAAV genomes for use in PCR amplification of large fragments. A primerwithin a conserved region located in the middle of the rep gene isutilized (AV1ns: 5′ GCTGCGTCAACTGGACCAATGAGAAC 3′, nt of SEQ ID NO:6) incombination with the 3′ primer located in another conserved regiondownstream of the Cap gene (AV2cas: 5′ CGCAGAGACCAAAGTTCAACTGAAACGA 3′,SEQ ID NO: 7) for amplification of AAV sequences including thefull-length AAV cap. Typically, following amplification, the productsare cloned and sequence analysis is performed with an accuracy of 99.9%.Using this method, the inventors have isolated at least 50 capsid cloneswhich have subsequently been characterized. Among them, 37 clones werederived from Rhesus macaque tissues (rh.1-rh.37), 6 clones fromcynomologous macaques (cy.1-cy.6), 2 clones from Baboons (bb.1and bb.2)and 5 clones from Chimps (ch.1-ch.5). These clones are identifiedelsewhere in the specification, together with the species of animal fromwhich they were identified and the tissues in that animal these novelsequences have been located.

C. Alternative Method for Isolating Novel AAV

In another aspect, the invention provides an alternative method forisolating novel AAV from a cell. This method involves infecting the cellwith a vector which provides helper functions to the AAV; isolatinginfectious clones containing AAV; sequencing the isolated AAV; andcomparing the sequences of the isolated AAV to known AAV serotypes,whereby differences in the sequences of the isolated AAV and known AAVserotypes indicates the presence of a novel AAV.

In one embodiment, the vector providing helper functions providesessential adenovirus functions, including, e.g., E1a, E1b, E2a, E4ORF6.In one embodiment, the helper functions are provided by an adenovirus.The adenovirus may be a wild-type adenovirus, and may be of human ornon-human origin, preferably non-human primate (NHP) origin. The DNAsequences of a number of adenovirus types are available from Genbank,including type Ad5 [Genbank Accession No. M73260]. The adenovirussequences may be obtained from any known adenovirus serotype, such asserotypes 2, 3, 4, 7, 12 and 40, and further including any of thepresently identified human types [see, e.g., Horwitz, cited above].Similarly adenoviruses known to infect non-human animals (e.g.,chimpanzees) may also be employed in the vector constructs of thisinvention. See, e.g., U.S. Pat. No. 6,083,716. In addition to wild-typeadenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids,episomes, etc.) carrying the necessary helper functions may be utilized.Such recombinant viruses are known in the art and may be preparedaccording to published techniques. See, e.g., U.S. Pat. No. 5,871,982and U.S. Pat. No. 6,251,677, which describe a hybrid Ad/AAV virus. Theselection of the adenovirus type is not anticipated to limit thefollowing invention. A variety of adenovirus strains are available fromthe American Type Culture Collection, Manassas, Va., or available byrequest from a variety of commercial and institutional sources. Further,the sequences of many such strains are available from a variety ofdatabases including, e.g., PubMed and GenBank.

In another alternative, infectious AAV may be isolated using genomewalking technology (Siebert et al., 1995, Nucleic Acid Research,23:1087-1088, Friezner-Degen et al., 1986, J Biol. Chem. 261:6972-6985,BD Biosciences Clontech, Palo Alto, Calif.). Genome walking isparticularly well suited for identifying and isolating the sequencesadjacent to the novel sequences identified according to the method ofthe invention. For example, this technique may be useful for isolatinginverted terminal repeat (ITRs) of the novel AAV serotype, based uponthe novel AAV capsid and/or rep sequences identified using the methodsof the invention. This technique is also useful for isolating sequencesadjacent to other AAV and non-AAV sequences identified and isolatedaccording to the present invention. See, Examples 3 and 4.

The methods of the invention may be readily used for a variety ofepidemiology studies, studies of biodistribution, monitoring of genetherapy via AAV vectors and vector derived from other integratedviruses. Thus, the methods are well suited for use in pre-packaged kitsfor use by clinicians, researchers, and epidemiologists.

II Diagnostic Kit

In another aspect, the invention provides a diagnostic kit for detectingthe presence of a known or unknown adeno-associated virus (AAV) in asample. Such a kit may contain a first set of 5′ and 3′ PCR primersspecific for a signature region of the AAV nucleic acid sequence.Alternatively, or additionally, such a kit can contain a first set of 5′and 3′ PCR primers specific for the 3.1 kb fragment which includes thefull-length AAV capsid nucleic acid sequence identified herein (e.g.,the AV1ns and AV2cas primers.) Optionally, a kit of the invention mayfurther contain two or more additional sets of 5′ and 3′ primers, asdescribed herein, and/or PCR probes. These primers and probes are usedaccording to the present invention amplify signature regions of each AAVserotype, e.g., using quantitative PCR.

The invention further provides a kit useful for identifying an AAVserotype detected according to the method of the invention and/or fordistinguishing novel AAV from known AAV. Such a kit may further includeone or more restriction enzymes, standards for AAV serotypes providingtheir “signature restriction enzyme digestions analyses”, and/or othermeans for determining the serotype of the AAV detected.

In addition, kits of the invention may include, instructions, a negativeand/or positive control, containers, diluents and buffers for thesample, indicator charts for signature comparisons, disposable gloves,decontamination instructions, applicator sticks or containers, andsample preparator cups, as well as any desired reagents, includingmedia, wash reagents and concentration reagents. Such reagents may bereadily selected from among the reagents described herein, and fromamong conventional concentration reagents. In one desirable embodiment,the wash reagent is an isotonic saline solution which has been bufferedto physiologic pH, such as phosphate buffered saline (PBS); the elutionreagent is PBS containing 0.4 M NaCl, and the concentration reagents anddevices. For example, one of skill in the art will recognize thatreagents such as polyethylene glycol (PEG), or NH₄SO₄ may be useful, orthat devices such as filter devices. For example, a filter device with a100 K membrane would concentrate rAAV.

The kits provided by the present invention are useful for performing themethods described herein, and for study of biodistribution,epidemiology, mode of transmission of novel AAV serotypes in human andNHPs.

Thus, the methods and kits of the invention permit detection,identification, and isolation of target viral sequences, particularlyintegrated viral sequences. The methods and kits are particularly wellsuited for use in detection, identification and isolation of AAVsequences, which may include novel AAV serotypes.

In one notable example, the method of the invention facilitated analysisof cloned AAV sequences by the inventors, which revealed heterogeneityof proviral sequences between cloned fragments from different animals,all of which were distinct from the known six AAV serotypes, with themajority of the variation localized to hypervariable regions of thecapsid protein. Surprising divergence of AAV sequences was noted inclones isolated from single tissue sources, such as lymph node, from anindividual rhesus monkey. This heterogeneity is best explained byapparent evolution of AAV sequence within individual animals due, inpart, to extensive homologous recombination between a limited number ofco-infecting parenteral viruses. These studies suggest sequenceevolution of widely disseminated virus during the course of a naturalAAV infection that presumably leads to the formation of swarms ofquasispecies which differ from one another in the array of capsidhypervariable regions. This is the first example of rapid molecularevolution of a DNA virus in a way that formerly was thought to berestricted to RNA viruses.

Sequences of several novel AAV serotypes identified by the method of theinvention and characterization of these serotypes is provided.

III. Novel AAV Serotypes

A. Nucleic Acid Sequences

Nucleic acid sequences of novel AAV serotypes identified by the methodsof the invention are provided. See, SEQ ID NO:1, 9-59, and 117-120,which are incorporated by reference herein. See also, FIG. 1 and thesequence listing.

For novel serotype AAV7, the full-length sequences, including the AAV 5′ITRs, capsid, rep, and AAV 3′ ITRs are provided in SEQ ID NO:1.

For other novel AAV serotypes of the invention, the approximately 3.1 kbfragment isolated according to the method of the invention is provided.This fragment contains sequences encoding full-length capsid protein andall or part of the sequences encoding the rep protein. These sequencesinclude the clones identified below.

For still other novel AAV serotypes, the signature region encoding thecapsid protein is provided. For example, the AAV10 nucleic acidsequences of the invention include those illustrated in FIG. 1 [See, SEQID NO:117, which spans 255 bases]. The AAV11 nucleic acid sequences ofthe invention include the DNA sequences illustrated in FIG. 1 [See, SEQID NO:118 which spans 258 bases]. The AAV12 nucleic acid sequences ofthe invention include the DNA sequences illustrated in FIG. 1 [See, SEQID NO:119, which consists of 255 bases ]. Using the methodologydescribed above, further AAV10, AAV11 and AAV12 sequences can be readilyidentified and used for a variety of purposes, including those describedfor AAV7 and the other novel serotypes herein.

FIG. 1 provides the non-human primate (NHP) AAV nucleic acid sequencesof the invention in an alignment with the previously published AAVserotypes, AAV 1 [SEQ ID NO:6], AAV2 [SEQ ID NO:7], and AAV3 [SEQ IDNO:8]. These novel

NHP sequences include those provided in the following Table I, which areidentified by clone number:

TABLE 1 AAV Cap Clone Source SEQ ID NO Sequence Number Species Tissue(DNA) Rh.1 Clone 9 Rhesus Heart 5 (AAV9) Rh.2 Clone 43.1 Rhesus MLN 39Rh.3 Clone 43.5 Rhesus MLN 40 Rh.4 Clone 43.12 Rhesus MLN 41 Rh.5 Clone43.20 Rhesus MLN 42 Rh.6 Clone 43.21 Rhesus MLN 43 Rh.7 Clone 43.23Rhesus MLN 44 Rh.8 Clone 43.25 Rhesus MLN 45 Rh.9 Clone 44.1 RhesusLiver 46 Rh.10 Clone 44.2 Rhesus Liver 59 Rh.11 Clone 44.5 Rhesus Liver47 Rh.12 Clone Rhesus MLN 30 42.1B Rh.13 42.2 Rhesus MLN 9 Rh.14 CloneRhesus MLN 32 42.3A Rh.15 Clone Rhesus MLN 36 42.3B Rh.16 Clone 42.4Rhesus MLN 33 Rh.17 Clone Rhesus MLN 34 42.5A Rh.18 Clone Rhesus MLN 2942.5B Rh.19 Clone Rhesus MLN 38 42.6B Rh.20 Clone 42.8 Rhesus MLN 27Rh.21 Clone 42.10 Rhesus MLN 35 Rh.22 Clone 42.11 Rhesus MLN 37 Rh.23Clone 42.12 Rhesus MLN 58 Rh.24 Clone 42.13 Rhesus MLN 31 Rh.25 Clone42.15 Rhesus MLN 28 Rh.26 Clone 223.2 Rhesus Liver 49 Rh.27 Clone 223.4Rhesus Liver 50 Rh.28 Clone 223.5 Rhesus Liver 51 Rh.29 Clone 223.6Rhesus Liver 52 Rh.30 Clone 223.7 Rhesus Liver 53 Rh.31 Clone RhesusLiver 48 223.10 Rh.32 Clone C1 Rhesus Spleen, Duo, 19 Kid & Liver Rh.33Clone C3 Rhesus 20 Rh.34 Clone C5 Rhesus 21 Rh.35 Clone F1 Rhesus Liver22 Rh.36 Clone F3 Rhesus 23 Rh.37 Clone F5 Rhesus 24 Cy.1 Clone 1.3 CynoBlood 14 Cy.2 Clone Cyno Blood 15 13.3B Cy.3 Clone 24.1 Cyno Blood 16Cy.4 Clone 27.3 Cyno Blood 17 Cy.5 Clone 7.2 Cyno Blood 18 Cy.6 Clone16.3 Cyno Blood 10 bb.1 Clone 29.3 Baboon Blood 11 bb.2 Clone 29.5Baboon Blood 13 Ch.1 Clone A3.3 Chimp Blood 57 Ch.2 Clone A3.4 ChimpBlood 54 Ch.3 Clone A3.5 Chimp Blood 55 Ch.4 Clone A3.7 Chimp Blood 56

A novel NHP clone was made by splicing capsids fragments of two chimpadenoviruses into an AAV2 rep construct. This new clone, A3.1, is alsotermed Ch.5 [SEQ ID NO:20]. Additionally, the present invention includestwo human AAV sequences, termed H6 [SEQ ID NO:25] and H2 [SEQ ID NO:26].

The AAV nucleic acid sequences of the invention further encompass thestrand which is complementary to the strands provided in the sequencesprovided in FIG. 1 and the Sequence Listing [SEQ ID NO:1, 9-59,117-120], nucleic acid sequences, as well as the RNA and cDNA sequencescorresponding to the sequences provided in FIG. 1 and the SequenceListing [SEQ ID NO:1, 9-59, 117-120], and their complementary strands.Also included in the nucleic acid sequences of the invention are naturalvariants and engineered modifications of the sequences of FIG. 1 and theSequence Listing [SEQ ID NO:1, 9-59, 117-120], and their complementarystrands. Such modifications include, for example, labels which are knownin the art, methylation, and substitution of one or more of thenaturally occurring nucleotides with a degenerate nucleotide.

Further included in this invention are nucleic acid sequences which aregreater than 85%, preferably at least about 90%, more preferably atleast about 95%, and most preferably at least about 98 to 99% identicalor homologous to the sequences of the invention, including FIG. 1 andthe Sequence Listing [SEQ ID NO:1, 9-59, 117-120]. These terms are asdefined herein.

Also included within the invention are fragments of the novel AAVsequences identified by the method described herein. Suitable fragmentsare at least 15 nucleotides in length, and encompass functionalfragments, i.e., fragments which are of biological interest. In oneembodiment, these fragments are fragments of the novel sequences of FIG.1 and the Sequence Listing [SEQ ID NO:1, 9-59, 117-120], theircomplementary strands, cDNA and RNA complementary thereto.

Examples of suitable fragments are provided with respect to the locationof these fragments on AAV1, AAV2, or AAV7. However, using the alignmentprovided herein (obtained using the Clustal W program at defaultsettings), or similar techniques for generating an alignment with othernovel serotypes of the invention, one of skill in the art can readilyidentify the precise nucleotide start and stop codons for desiredfragments.

Examples of suitable fragments include the sequences encoding the threevariable proteins (vp) of the AAV capsid which are alternative splicevariants: vp1 [e.g., nt 825 to 3049 of AAV7, SEQ ID NO: 1]; vp2 [e.g.,nt 1234-3049 of AAV7, SEQ ID NO: 1]; and vp 3 [e.g., nt 1434-3049 ofAAV7, SEQ ID NO:11. It is notable that AAV7 has an unusual GTG startcodon. With the exception of a few house-keeping genes, such a startcodon has not previously been reported in DNA viruses. The start codonsfor vp1, vp2 and vp3 for other AAV serotypes have been believed to besuch that they permit the cellular mechanism of the host cell in whichthey reside to produce vp1, vp2 and vp3 in a ratio of 10%:10%:80%,respectively, in order to permit efficient assembly of the virion.However, the AAV7 virion has been found to assemble efficiently evenwith this rare GTG start codon. Thus, the inventors anticipate this itis desirable to alter the start codon of the vp3 of other AAV serotypesto contain this rare GTG start codon, in order to improve packagingefficiency, to alter the virion structure and/or to alter location ofepitopes (e.g., neutralizing antibody epitopes) of other AAV serotypes.The start codons may be altered using conventional techniques including,e.g., site directed mutagenesis. Thus, the present invention encompassesaltered AAV virions of any selected serotype, composed of a vp 3, and/oroptionally, vp1 and/or vp2 having start codons altered to GTG.

Other suitable fragments of AAV, include a fragment containing the startcodon for the AAV capsid protein [e.g., nt 468 to 3090 of AAV7, SEQ IDNO:1, nt 725 to 3090 of AAV7, SEQ ID NO: 1, and corresponding regions ofthe other AAV serotypes]. Still other fragments of AAV7 and the othernovel AAV serotypes identified using the methods described hereininclude those encoding the rep proteins, including rep 78 [e.g.,initiation codon 334 of FIG. 1 for AAV7], rep 68 [initiation codon nt334 of FIG. 1 for AAV7], rep 52 [initiation codon 1006 of FIG. 1 forAAV7], and rep 40 [initiation codon 1006 of FIG. 1 for AAV7] Otherfragments of interest may include the AAV 5′ inverted terminal repeatsITRs, [nt 1 to 107 of FIG. 1 for AAV7]; the AAV 3′ ITRs [nt 4704 to 4721of FIG. 1 for AAV7], P19 sequences, AAV P40 sequences, the rep bindingsite, and the terminal resolute site (TRS). Still other suitablefragments will be readily apparent to those of skill in the art. Thecorresponding regions in the other novel serotypes of the invention canbe readily determined by reference to FIG. 1, or by utilizingconventional alignment techniques with the sequences provided herein.

In addition to including the nucleic acid sequences provided in thefigures and Sequence Listing, the present invention includes nucleicacid molecules and sequences which are designed to express the aminoacid sequences, proteins and peptides of the AAV serotypes of theinvention. Thus, the invention includes nucleic acid sequences whichencode the following novel AAV amino acid sequences: C1 [SEQ ID NO:60],C2 [SEQ ID NO:61], C5 [SEQ ID NO:62], A3-3 [SEQ ID NO:66], A3-7 [SEQ IDNO:67], A3-4 [SEQ ID NO:68], A3-5 [SEQ ID NO: 69], 3.3b [SEQ ID NO: 62],223.4 [SEQ ID NO: 73], 223-5 [SEQ ID NO:74], 223-10 [SEQ ID NO:75],223-2 [SEQ ID NO:76], 223-7 [SEQ ID NO: 77], 223-6 [SEQ ID NO: 78], 44-1[SEQ ID NO: 79], 44-5 [SEQ ID NO:80], 44-2 [SEQ ID NO:81], 42-15 [SEQ IDNO: 84], 42-8 [SEQ ID NO: 85], 42-13 [SEQ ID NO:86], 42-3A [SEQ IDNO:87], 42-4 [SEQ ID NO:88], 42-5A [SEQ ID NO:89], 42-1B [SEQ ID NO:90],42-5B [SEQ ID NO:91], 43-1 [SEQ ID NO: 92], 43-12 [SEQ ID NO: 93], 43-5[SEQ ID NO:94], 43-21 [SEQ ID NO:96], 43-25 [SEQ ID NO: 97], 43-20 [SEQID NO:99], 24.1 [SEQ ID NO: 101], 42.2 [SEQ ID NO:102], 7.2 [SEQ ID NO:103], 27.3 [SEQ ID NO: 104], 16.3 [SEQ ID NO: 105], 42.10 [SEQ ID NO:106], 42-3B [SEQ ID NO: 107], 42-11 [SEQ ID NO: 108], F1 [SEQ ID NO:109], F5 [SEQ ID NO: 110], F3 [SEQ ID NO:111], 42-6B [SEQ ID NO: 112],and/or 42-12 [SEQ ID NO: 113], and artificial AAV serotypes generatedusing these sequences and/or unique fragments thereof.

As used herein, artificial AAV serotypes include, without limitation,AAV with a non-naturally occurring capsid protein. Such an artificialcapsid may be generated by any suitable technique, using a novel AAVsequence of the invention (e.g., a fragment of a vp1 capsid protein) incombination with heterologous sequences which may be obtained fromanother AAV serotype (known or novel), non-contiguous portions of thesame AAV serotype, from a non-AAV viral source, or from a non-viralsource. An artificial AAV serotype may be, without limitation, achimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAVcapsid.

B. AAV Amino Acid Sequences, Proteins and Peptides

The invention provides proteins and fragments thereof which are encodedby the nucleic acid sequences of the novel AAV serotypes identifiedherein, including, e.g., AAV7 [nt 825 to 3049 of AAV7, SEQ ID NO: 1] theother novel serotypes provided herein. Thus, the capsid proteins of thenovel serotypes of the invention, including: H6 [SEQ ID NO: 25], H2 [SEQID NO: 26], 42-2 [SEQ ID NO:9], 42-8 [SEQ ID NO:27], 42-15 [SEQ IDNO:28], 42-5b [SEQ ID NO: 29], 42-1b [SEQ ID NO:30]; 42-13 [SEQ ID NO:31], 42-3a [SEQ ID NO: 32], 42-4 [SEQ ID NO:33], 42-5a [SEQ ID NO: 34],42-10 [SEQ ID NO:35], 42-3b [SEQ ID NO: 36], 42-11 [SEQ ID NO: 37],42-6b [SEQ ID NO:38], 43-1 [SEQ ID NO: 39], 43-5 [SEQ ID NO: 40], 43-12[SEQ ID NO:41], 43-20 [SEQ ID NO:42], 43-21 [SEQ ID NO: 43], 43-23 [SEQID NO:44], 43-25 [SEQ ID NO: 45], 44.1 [SEQ ID NO:47], 44.5 [SEQ IDNO:47], 223.10 [SEQ ID NO:48], 223.2 [SEQ ID NO:49], 223.4 [SEQ IDNO:50], 223.5 [SEQ ID NO: 51], 223.6 [SEQ ID NO: 52], 223.7 [SEQ ID NO:53], A3.4 [SEQ ID NO: 54], A3.5 [SEQ ID NO:55], A3.7 [SEQ ID NO: 56],A3.3 [SEQ ID NO:57], 42.12 [SEQ ID NO: 58], and 44.2 [SEQ ID NO: 59],can be readily generated using conventional techniques from the openreading frames provided for the above-listed clones.

The invention further encompasses AAV serotypes generated usingsequences of the novel AAV serotypes of the invention, which aregenerated using synthetic, recombinant or other techniques known tothose of skill in the art. The invention is not limited to novel AAVamino acid sequences, peptides and proteins expressed from the novel AAVnucleic acid sequences of the invention and encompasses amino acidsequences, peptides and proteins generated by other methods known in theart, including, e.g., by chemical synthesis, by other synthetictechniques, or by other methods. For example, the sequences of any of C1[SEQ ID NO:60], C2 [SEQ ID NO:61], C5 [SEQ ID NO:62], A3-3 [SEQ IDNO:66], A3-7 [SEQ ID NO:67], A3-4 [SEQ ID NO:68], A3-5 [SEQ ID NO: 69],3.3b [SEQ ID NO: 62], 223.4 [SEQ ID NO: 73], 223-5 [SEQ ID NO:74],223-10 [SEQ ID NO:75], 223-2 [SEQ ID NO:76], 223-7 [SEQ ID NO: 77],223-6 [SEQ ID NO: 78], 44-1 [SEQ ID NO: 79], 44-5 [SEQ ID NO:80], 44-2[SEQ ID NO:81], 42-15 [SEQ ID NO: 84], 42-8 [SEQ ID NO: 85], 42-13 [SEQID NO:86], 42-3A [SEQ ID NO:87], 42-4 [SEQ ID NO:881, 42-5A [SEQ IDNO:89], 42-1B [SEQ ID NO:90], 42-5B [SEQ ID NO:91], 43-1 [SEQ ID NO:92], 43-12 [SEQ ID NO: 93], 43-5 [SEQ ID NO:94], 43-21 [SEQ ID NO:96],43-25 [SEQ ID NO: 97], 43-20 [SEQ ID NO:99], 24.1 [SEQ ID NO: 101], 42.2[SEQ ID NO:102], 7.2 [SEQ ID NO: 103], 27.3 [SEQ ID NO: 104], 16.3 [SEQID NO: 105], 42.10 [SEQ ID NO: 106], 42-3B [SEQ ID NO: 107], 42-11 [SEQID NO: 108], F1 [SEQ ID NO: 109], F5 [SEQ ID NO: 110], F3 [SEQ IDNO:111], 42-6B [SEQ ID NO: 112], and/or 42-12 [SEQ ID NO: 113] by bereadily generated using a variety of techniques.

Suitable production techniques are well known to those of skill in theart. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively,peptides can also be synthesized by the well known solid phase peptidesynthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962);Stewart and Young, Solid Phase Peptide Synthesis (Freeman, SanFrancisco, 1969) pp. 27-62). These and other suitable production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

Particularly desirable proteins include the AAV capsid proteins, whichare encoded by the nucleotide sequences identified above. The sequencesof many of the capsid proteins of the invention are provided in analignment in FIG. 2 and/or in the Sequence Listing, SEQ ID NO: 2 and 60to 115, which is incorporated by reference herein. The AAV capsid iscomposed of three proteins, vp1, vp2 and vp3, which are alternativesplice variants. The full-length sequence provided in these figures isthat of vp1. Based on the numbering of the AAV7 capsid [SEQ ID NO:2],the sequences of vp2 span amino acid 138-737 of AAV7 and the sequencesof vp3 span amino acids 203-737 of AAV7. With this information, one ofskill in the art can readily determine the location of the vp2 and vp3proteins for the other novel serotypes of the invention.

Other desirable proteins and fragments of the capsid protein include theconstant and variable regions, located between hypervariable regions(HPV) and the sequences of the HPV regions themselves. An algorithmdeveloped to determine areas of sequence divergence in AAV2 has yielded12 hypervariable regions (HVR) of which 5 overlap or are part of thefour previously described variable regions. [Chiorini et al, J. Virol,73:1309-19 (1999); Rutledge et al, J Virol., 72:309-319] Using thisalgorithm and/or the alignment techniques described herein, the HVR ofthe novel AAV serotypes are determined. For example, with respect to thenumber of the AAV2 vp1 [SEQ ID NO:70], the HVR are located as follows:HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264; HVR4, aa 381-383;HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa500-504; HVR8, aa 514-522;HVR9, aa 534-555; HVR10, aa 581-594; HVR11, aa 658-667; and HVR12, aa705-719. Utilizing an alignment prepared in accordance with conventionalmethods and the novel sequences provided herein [See, e.g., FIG. 2], onecan readily determine the location of the HVR in the novel AAV serotypesof the invention. For example, utilizing FIG. 2, one can readilydetermine that for AAV7 [SEQ ID NO:2]. HVR1 is located at aa 146-152;HVR2 is located at 182-187; HVR3 is located at aa 263-266, HVR4 islocated at aa 383-385, HVR5 is located at aa 451-475; HVR6 is located ataa 491-496 of AAV7; HVR7 is located at aa 501-505; HVR8 is located at aa513-521; HVR9 is located at 533-554; HVR10 is located at aa 583-596;HVR11 is located at aa 660-669; HVR12 is located at aa 707-721. Usingthe information provided herein, the HVRs for the other novel serotypesof the invention can be readily determined.

In addition, within the capsid, amino acid cassettes of identity havebeen identified. These cassettes are of particular interest, as they areuseful in constructing artificial serotypes, e.g., by replacing a HVR1cassette of a selected serotype with an HVR1 cassette of anotherserotype. Certain of these cassettes of identity are noted in FIG. 2.See, FIG. 2, providing the Clustal X alignment, which has a ruler isdisplayed below the sequences, starting at 1 for the first residueposition. The line above the ruler is used to mark strongly conservedpositions. Three characters (*, :, .) are used. “*” indicates positionswhich have a single, fully conserved residue. “:” indicates that a“strong” group is fully conserved “.” Indicates that a “weaker” group isfully conserved. These are all the positively scoring groups that occurin the Gonnet Pam250 matrix. The strong groups are defined as a strongscore >0.5 and the weak groups are defined as weak score <0.5.

Additionally, examples of other suitable fragments of AAV capsidsinclude, with respect to the numbering of AAV2 [SEQ ID NO:70], aa 24-42,aa 25-28; aa 81-85; aa133-165; aa 134-165; aa 137-143; aa 154-156; aa194-208; aa 261-274; aa 262-274; aa 171-173; aa 413-417; aa 449-478; aa494-525; aa 534-571; aa 581-601; aa 660-671; aa 709-723. Still otherdesirable fragments include, for example, in AAV7, amino acids 1 to 184of SEQ ID NO:2, amino acids 199 to 259; amino acids 274 to 446; aminoacids 603 to 659; amino acids 670 to 706; amino acids 724 to 736; aa 185to 198; aa 260 to 273; aa447 to 477; aa495 to 602; aa660 to 669; andaa707 to 723. Still other desirable regions, based on the numbering ofAAV7 [SEQ ID NO:2], are selected from among the group consisting of aa185 to 198; aa 260 to 273; aa447 to 477; aa495 to 602; aa660 to 669; andaa707 to 723. Using the alignment provided herein performed using theClustal X program at default settings, or using other commercially orpublicly available alignment programs at default settings, one of skillin the art can readily determine corresponding fragments of the novelAAV capsids of the invention.

Other desirable proteins are the AAV rep proteins [aa 1 to 623 of SEQ IDNO:3 for AAV7] and functional fragments thereof, including, e.g., aa 1to 171, aa 172 to 372, aa 373 to 444, aa 445 to 623 of SEQ ID NO:3,among others. Suitably, such fragments are at least 8 amino acids inlength. See, FIG. 3. Comparable regions can be identified in theproteins of the other novel AAV of the invention, using the techniquesdescribed herein and those which are known in the art. In addition,fragments of other desired lengths may be readily utilized. Suchfragments may be produced recombinantly or by other suitable means,e.g., chemical synthesis.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Such production methods are withinthe knowledge of those of skill in the art and are not a limitation ofthe present invention.

IV. Production of rAAV with Novel AAV Capsids

The invention encompasses novel, wild-type AAV serotypes identified bythe invention, the sequences of which wild-type AAV serotypes are freeof DNA and/or cellular material with these viruses are associated innature. In another aspect, the present invention provides moleculeswhich utilize the novel AAV sequences of the invention, includingfragments thereof, for production of molecules useful in delivery of aheterologous gene or other nucleic acid sequences to a target cell.

The molecules of the invention which contain sequences of a novel AAVserotype of the invention include any genetic element (vector) which maybe delivered to a host cell, e.g., naked DNA, a plasmid, phage,transposon, cosmid, episome, a protein in a non-viral delivery vehicle(e.g., a lipid-based carrier), virus, etc. which transfer the sequencescarried thereon. The selected vector may be delivered by any suitablemethod, including transfection, electroporation, liposome delivery,membrane fusion techniques, high velocity DNA-coated pellets, viralinfection and protoplast fusion. The methods used to construct anyembodiment of this invention are known to those with skill in nucleicacid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Sambrook et al,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.

In one embodiment, the vectors of the invention contain sequencesencoding a novel AAV capsid of the invention (e.g., AAV7 capsid, AAV44-2 (rh.10), an AAV10 capsid, an AAV11 capsid, an AAV12 capsid), or afragment of one or more of these AAV capsids. Alternatively, the vectorsmay contain the capsid protein, or a fragment thereof, itself.

Optionally, vectors of the invention may contain sequences encoding AAVrep proteins. Such rep sequences may be from the same AAV serotype whichis providing the cap sequences. Alternatively, the present inventionprovides vectors in which the rep sequences are from an AAV serotypewhich differs from that which is providing the cap sequences. In oneembodiment, the rep and cap sequences are expressed from separatesources (e.g., separate vectors, or a host cell and a vector). Inanother embodiment, these rep sequences are expressed from the samesource as the cap sequences. In this embodiment, the rep sequences maybe fused in frame to cap sequences of a different AAV serotype to form achimeric AAV vector. Optionally, the vectors of the invention furthercontain a minigene comprising a selected transgene which is flanked byAAV 5′ ITR and AAV 3′ ITR.

Thus, in one embodiment, the vectors described herein contain nucleicacid sequences encoding an intact AAV capsid which may be from a singleAAV serotype (e.g., AAV7 or another novel AAV). Alternatively, thesevectors contain sequences encoding artificial capsids which contain oneor more fragments of the AAV7 (or another novel AAV) capsid fused toheterologous AAV or non-AAV capsid proteins (or fragments thereof).These artificial capsid proteins are selected from non-contiguousportions of the AAV7 (or another novel AAV) capsid or from capsids ofother AAV serotypes. For example, it may be desirable to modify thecoding regions of one or more of the AAV vp1, e.g., in one or more ofthe hypervariable regions (i.e., HPV1-12), or vp2, and/or vp3. Inanother example, it may be desirable to alter the start codon of the vp3protein to GTG. These modifications may be to increase expression,yield, and/or to improve purification in the selected expressionsystems, or for another desired purpose (e.g., to change tropism oralter neutralizing antibody epitopes).

The vectors described herein, e.g., a plasmid, are useful for a varietyof purposes, but are particularly well suited for use in production of arAAV containing a capsid comprising AAV sequences or a fragment thereof.These vectors, including rAAV, their elements, construction, and usesare described in detail herein.

In one aspect, the invention provides a method of generating arecombinant adeno-associated virus (AAV) having an AAV serotype 7 (oranother novel AAV) capsid, or a portion thereof. Such a method involvesculturing a host cell which contains a nucleic acid sequence encoding anadeno-associated virus (AAV) serotype 7 (or another novel AAV) capsidprotein, or fragment thereof, as defined herein; a functional rep gene;a minigene composed of, at a minimum, AAV inverted terminal repeats(ITRs) and a transgene; and sufficient helper functions to permitpackaging of the minigene into the AAV7 (or another novel AAV) capsidprotein.

The components required to be cultured in the host cell to package anAAV minigene in an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e g ,minigene, rep sequences, cap sequences, and/or helper functions) may beprovided by a stable host cell which has been engineered to contain oneor more of the required components using methods known to those of skillin the art. Most suitably, such a stable host cell will contain therequired component(s) under the control of an inducible promoter.However, the required component(s) may be under the control of aconstitutive promoter. Examples of suitable inducible and constitutivepromoters are provided herein, in the discussion of regulatory elementssuitable for use with the transgene. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated which is derived from 293 cells (whichcontain E 1 helper functions under the control of a constitutivepromoter), but which contains the rep and/or cap proteins under thecontrol of inducible promoters. Still other stable host cells may begenerated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transferthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

A. The Minigene

The minigene is composed of, at a minimum, a transgene and itsregulatory sequences, and 5= and 3=AAV inverted terminal repeats (ITRs).It is this minigene which is packaged into a capsid protein anddelivered to a selected host cell.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc.

These coding sequences, when associated with regulatory elements whichdrive their expression, provide signals detectable by conventionalmeans, including enzymatic, radiographic, colorimetric, fluorescence orother spectrographic assays, fluorescent activating cell sorting assaysand immunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for beta-galactosidaseactivity. Where the transgene is green fluorescent protein orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

However, desirably, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.One example of a useful RNA sequence is a sequence which extinguishesexpression of a targeted nucleic acid sequence in the treated animal

The transgene may be used to correct or ameliorate gene deficiencies,which may include deficiencies in which normal genes are expressed atless than normal levels or deficiencies in which the functional geneproduct is not expressed. A preferred type of transgene sequence encodesa therapeutic protein or polypeptide which is expressed in a host cell.The invention further includes using multiple transgenes, e.g., tocorrect or ameliorate a gene defect caused by a multi-subunit protein.In certain situations, a different transgene may be used to encode eachsubunit of a protein, or to encode different peptides or proteins. Thisis desirable when the size of the DNA encoding the protein subunit islarge, e.g., for an immunoglobulin, the platelet-derived growth factor,or a dystrophin protein. In order for the cell to produce themulti-subunit protein, a cell is infected with the recombinant viruscontaining each of the different subunits. Alternatively, differentsubunits of a protein may be encoded by the same transgene. In thiscase, a single transgene includes the DNA encoding each of the subunits,with the DNA for each subunit separated by an internal ribozyme entrysite (IRES). This is desirable when the size of the DNA encoding each ofthe subunits is small, e.g., the total size of the DNA encoding thesubunits and the IRES is less than five kilobases. As an alternative toan IRES, the DNA may be separated by sequences encoding a 2A peptide,which self-cleaves in a post-translational event. See, e.g., M. L.Donnelly, et al, J. Gen. Virol., 78(Pt 1):13-21 (Jan 1997); Furler, S.,et al, Gene Ther., 8(11):864-873 (June 2001); Klump H., et al., GeneTher., 8(10):811-817 (May 2001). This 2A peptide is significantlysmaller than an IRES, making it well suited for use when space is alimiting factor. However, the selected transgene may encode anybiologically active product or other product, e.g., a product desirablefor study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis invention.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements necessary which areoperably linked to the transgene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, Aoperably linked≅ sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system [WO 98/10088]; theecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)1, the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J Clin. Invest., 100:2865-2872(1997)]. Still other types of inducible promoters which may be useful inthis context are those which are regulated by a specific physiologicalstate, e.g., temperature, acute phase, a particular differentiationstate of the cell, or in replicating cells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally-occurringpromoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor α chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, plasmids carrying therapeutically useful transgenes may alsoinclude selectable markers or reporter genes may include sequencesencoding geneticin, hygromicin or purimycin resistance, among others.Such selectable reporters or marker genes (preferably located outsidethe viral genome to be rescued by the method of the invention) can beused to signal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the plasmid may include anorigin of replication. Selection of these and other promoters and vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The combination of the transgene, promoter/enhancer, and 5= and 3=ITRsis referred to as a “minigene” for ease of reference herein. Providedwith the teachings of this invention, the design of such a minigene canbe made by resort to conventional techniques.

3. Delivery of the Minigene to a Packaging Host Cell

The minigene can be carried on any suitable vector, e.g., a plasmid,which is delivered to a host cell. The plasmids useful in this inventionmay be engineered such that they are suitable for replication and,optionally, integration in prokaryotic cells, mammalian cells, or both.These plasmids (or other vectors carrying the 5′ AAV ITR-heterologousmolecule-3′ITR) contain sequences permitting replication of the minigenein eukaryotes and/or prokaryotes and selection markers for thesesystems. Selectable markers or reporter genes may include sequencesencoding geneticin, hygromicin or purimycin resistance, among others.The plasmids may also contain certain selectable reporters or markergenes that can be used to signal the presence of the vector in bacterialcells, such as ampicillin resistance. Other components of the plasmidmay include an origin of replication and an amplicon, such as theamplicon system employing the Epstein Barr virus nuclear antigen. Thisamplicon system, or other similar amplicon components permit high copyepisomal replication in the cells. Preferably, the molecule carrying theminigene is transfected into the cell, where it may exist transiently.Alternatively, the minigene (carrying the 5′ AAV ITR-heterologousmolecule-3′ ITR) may be stably integrated into the genome of the hostcell, either chromosomally or as an episome. In certain embodiments, theminigene may be present in multiple copies, optionally in head-to-head,head-to-tail, or tail-to-tail concatamers. Suitable transfectiontechniques are known and may readily be utilized to deliver the minigeneto the host cell.

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

B. Rep and Cap Sequences

In addition to the minigene, the host cell contains the sequences whichdrive expression of the novel AAV capsid protein (e.g., AAV7 or othernovel AAV capsid or an artificial capsid protein comprising a fragmentof one or more of these capsids) in the host cell and rep sequences ofthe same serotype as the serotype of the AAV ITRs found in the minigene.The AAV cap and rep sequences may be independently obtained from an AAVsource as described above and may be introduced into the host cell inany manner known to one in the art as described above. Additionally,when pseudotyping a novel AAV capsid of the invention, the sequencesencoding each of the essential rep proteins may be supplied by the sameAAV serotype, or the sequences encoding the rep proteins may be suppliedby different AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, orone of the novel serotypes identified herein). For example, the rep78/68sequences may be from AAV2, whereas the rep52/40 sequences may fromAAV1.

In one embodiment, the host cell stably contains the capsid proteinunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the capsid protein is expressedunder the control of an inducible promoter. In another embodiment, thecapsid protein is supplied to the host cell in trans. When delivered tothe host cell in trans, the capsid protein may be delivered via aplasmid which contains the sequences necessary to direct expression ofthe selected capsid protein in the host cell. Most desirably, whendelivered to the host cell in trans, the plasmid carrying the capsidprotein also carries other sequences required for packaging the rAAV,e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell. Mostdesirably, when delivered to the host cell in trans, the plasmidcarrying the capsid protein also carries other sequences required forpackaging the rAAV, e.g., the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the genome of the cell. Another embodimenthas the rep and cap sequences transiently expressed in the host cell.For example, a useful nucleic acid molecule for such transfectioncomprises, from 5′ to 3′, a promoter, an optional spacer interposedbetween the promoter and the start site of the rep gene sequence, an AAVrep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the minigene. The vector may comprise one or more of thegenes encoding the helper functions, e.g., the adenoviral proteins E1,E2a, and E4ORF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In one embodiment, an AAV P5 promotersequence is employed. The selection of the AAV to provide any of thesesequences does not limit the invention.

In another preferred embodiment, the promoter for rep is an induciblepromoter, as are discussed above in connection with the transgeneregulatory elements. One preferred promoter for rep expression is the T7promoter. The vector comprising the rep gene regulated by the T7promoter and the cap gene, is transfected or transformed into a cellwhich either constitutively or inducibly expresses the T7 polymerase.See WO 98/10088, published Mar. 12, 1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the phage laddersequences or yeast ladder sequences, which are available commercially,e.g., from Gibco or Invitrogen, among others. The spacer may be of anysize sufficient to reduce expression of the rep78 and rep68 geneproducts, leaving the rep52, rep40 and cap gene products expressed atnormal levels. The length of the spacer may therefore range from about10 bp to about 10.0 kbp, preferably in the range of about 100 bp toabout 8.0 kbp. To reduce the possibility of recombination, the spacer ispreferably less than 2 kbp in length; however, the invention is not solimited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions may besupplied by a herpesvirus. Most desirably, the necessary helperfunctions are each provided from a human or non-human primate adenovirussource, such as those described above and/or are available from avariety of sources, including the American Type Culture Collection(ATCC), Manassas, Va. (US). In one currently preferred embodiment, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Thehost cell may contain other adenoviral genes such as VAI RNA, but thesegenes are not required. In a preferred embodiment, no other adenovirusgenes or gene functions are present in the host cell.

By Aadenoviral DNA which expresses the E1a gene product≅, it is meantany adenovirus sequence encoding E1a or any functional E1a portion.Adenoviral DNA which expresses the E2a gene product and adenoviral DNAwhich expresses the E4 ORF6 gene products are defined similarly. Alsoincluded are any alleles or other modifications of the adenoviral geneor functional portion thereof. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the adenoviral function in some manner, as well asnaturally occurring allelic variants thereof. Such modifications andmethods for manipulating DNA to achieve the se adenovirus gene functionsare known to those of skill in the art.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by exogenously-added factors, forexample

D. Host Cells And Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The most desirable cellsdo not carry any adenovirus gene other than E 1, E2a and/or E4 ORF6; nordo they contain any other virus gene which could result in homologousrecombination of a contaminating virus during the production of rAAV;and it is capable of infection or transfection of DNA and expression ofthe transfected DNA. In a preferred embodiment, the host cell is onethat has rep and cap stably transfected in the cell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the minigene as described above. Stable rep and/or capexpressing cell lines, such as B-50 (PCT/US98/19463), or those describedin U.S. Pat. No. 5,658,785, may also be similarly employed. Anotherdesirable host cell contains the minimum adenoviral DNA which issufficient to express E4 ORF6. Yet other cell lines can be constructedusing the novel AAV rep and/or novel AAV cap sequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

These novel AAV-based vectors which are generated by one of skill in theart are beneficial for gene delivery to selected host cells and genetherapy patients since no neutralization antibodies to AAV7 have beenfound in the human population. Further, early studies show noneutralizing antibodies in cyno monkey and chimpanzee populations, andless than 15% cross-reactivity of AAV 7 in rhesus monkeys, the speciesfrom which the serotype was isolated. One of skill in the art mayreadily prepare other rAAV viral vectors containing the AAV7 capsidproteins provided herein using a variety of techniques known to those ofskill in the art. One may similarly prepare still other rAAV viralvectors containing AAV7 sequence and AAV capsids of another serotype.Similar advantages are conferred by the vectors based on the other novelAAV of the invention.

Thus, one of skill in the art will readily understand that the AAV7sequences of the invention can be readily adapted for use in these andother viral vector systems for in vitro, ex vivo or in vivo genedelivery. Similarly, one of skill in the art can readily select otherfragments of the novel AAV genome of the invention for use in a varietyof rAAV and non-rAAV vector systems. Such vectors systems may include,e.g., lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadenoviral systems, among others. Selection of these vector systems isnot a limitation of the present invention.

Thus, the invention further provides vectors generated using the nucleicacid and amino acid sequences of the novel AAV of the invention. Suchvectors are useful for a variety of purposes, including for delivery oftherapeutic molecules and for use in vaccine regimens. Particularlydesirable for delivery of therapeutic molecules are recombinant AAVcontaining capsids of the novel AAV of the invention. These, or othervector constructs containing novel AAV sequences of the invention may beused in vaccine regimens, e.g., for co-delivery of a cytokine, or fordelivery of the immunogen itself

V. Recombinant Viruses And Uses Thereof

Using the techniques described herein, one of skill in the art maygenerate a rAAV having a capsid of a novel serotype of the invention, ora novel capsid containing one or more novel fragments of an AAV serotypeidentified by the method of the invention. In one embodiment, afull-length capsid from a single serotype, e.g., AAV7 [SEQ ID NO: 2] canbe utilized. In another embodiment, a full-length capsid may begenerated which contains one or more fragments of a novel serotype ofthe invention fused in frame with sequences from another selected AAVserotype. For example, a rAAV may contain one or more of the novelhypervariable region sequences of an AAV serotype of the invention.Alternatively, the unique AAV serotypes of the invention may be used inconstructs containing other viral or non-viral sequences.

It will be readily apparent to one of skill in the art one embodiment,that certain serotypes of the invention will be particularly well suitedfor certain uses. For example, vectors based on AAV7 capsids of theinvention are particularly well suited for use in muscle; whereasvectors based on rh.10 (44-2) capsids of the invention are particularlywell suited for use in lung. Uses of such vectors are not so limited andone of skill in the art may utilize these vectors for delivery to othercell types, tissues or organs. Further, vectors based upon other capsidsof the invention may be used for delivery to these or other cells,tissues or organs.

A. Delivery of Transgene

In another aspect, the present invention provides a method for deliveryof a transgene to a host which involves transfecting or infecting aselected host cell with a vector generated with the sequences of the AAVof the invention. Methods for delivery are well known to those of skillin the art and are not a limitation of the present invention.

In one desirable embodiment, the invention provides a method forAAV-mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequenceswhich direct expression thereof and AAV capsid proteins.

Optionally, a sample from the host may be first assayed for the presenceof antibodies to a selected AAV serotype. A variety of assay formats fordetecting neutralizing antibodies are well known to those of skill inthe art. The selection of such an assay is not a limitation of thepresent invention. See, e.g., Fisher et al, Nature Med., 3(3):306-312(March 1997) and W. C. Manning et al, Human Gene Therapy, 9:477-485(Mar. 1, 1998). The results of this assay may be used to determine whichAAV vector containing capsid proteins of a particular serotype arepreferred for delivery, e.g., by the absence of neutralizing antibodiesspecific for that capsid serotype.

In one aspect of this method, the delivery of vector with a selected AAVcapsid proteins may precede or follow delivery of a gene via a vectorwith a different serotype AAV capsid protein. Similarly, the delivery ofvector with other novel AAV capsid proteins of the invention may precedeor follow delivery of a gene via a vector with a different serotype AAVcapsid protein. Thus, gene delivery via rAAV vectors may be used forrepeat gene delivery to a selected host cell. Desirably, subsequentlyadministered rAAV vectors carry the same transgene as the first rAAVvector, but the subsequently administered vectors contain capsidproteins of serotypes which differ from the first vector. For example,if a first vector has AAV7 capsid proteins [SEQ ID NO:21, subsequentlyadministered vectors may have capsid proteins selected from among theother serotypes, including AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV6, AAV10,AAV11, and AAV12, or any of the other novel AAV capsids identifiedherein including, without limitation: A3.1, H2, H6, C1, C2, C5, A3-3,A3-7, A3-4, A3-5, 3.3b, 223.4, 223-5, 223-10, 223-2, 223-7, 223-6, 44-1,44-5, 44-2, 42-15, 42-8, 42-13, 42-3A, 42-4, 42-5A, 42-1B, 42-5B, 43-1,43-12, 43-5, 43-21, 43-25, 43-20, 24.1, 42.2, 7.2, 27.3, 16.3, 42.10,42-3B, 42-11, F1, F5, F3, 42-6B, and/or 42-12.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian patient. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin

The viral vectors are administered in sufficient amounts to transfectthe cells and to provide sufficient levels of gene transfer andexpression to provide a therapeutic benefit without undue adverseeffects, or with medically acceptable physiological effects, which canbe determined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, and other parental routes of administration.Routes of administration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about1 ml to about 100 ml of solution containing concentrations of from about1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage may beabout 1×10¹³ to 1×10¹⁶ AAV genomes. The dosage will be adjusted tobalance the therapeutic benefit against any side effects and suchdosages may vary depending upon the therapeutic application for whichthe recombinant vector is employed. The levels of expression of thetransgene can be monitored to determine the frequency of dosageresulting in viral vectors, preferably AAV vectors containing theminigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention.

Examples of therapeutic products and immunogenic products for deliveryby the AAV-containing vectors of the invention are provided below. Thesevectors may be used for a variety of therapeutic or vaccinal regimens,as described herein. Additionally, these vectors may be delivered incombination with one or more other vectors or active ingredients in adesired therapeutic and/or vaccinal regimen.

B. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor α (TGFα), platelet-derived growthfactor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), anyone of the transforming growth factor β superfamily, including TGFβ,activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs1-15, any one of the heregluin/neuregulin/ARIA/neu differentiationfactor (NDF) family of growth factors, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5,ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophicfactor (GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, IL-2, IL-4, IL-12, and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors a and β,interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, and the scavenger receptor. The inventionalso encompasses gene products such as members of the steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include transcription factors such as jun, fos,max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD andmyogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence. Still other useful gene products includeenzymes such as may be useful in enzyme replacement therapy, which isuseful in a variety of conditions resulting from deficient activity ofenzyme. For example, enzymes that contain mannose-6-phosphate may beutilized in therapies for lysosomal storage diseases (e.g., a suitablegene includes that encoding β-glucuronidase (GUSB)).

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce Aself≅-directed antibodies. Tcell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors (TCRs) that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases.

C. Immunogenic Transgenes

Alternatively, or in addition, the vectors of the invention may containAAV sequences of the invention and a transgene encoding a peptide,polypeptide or protein which induces an immune response to a selectedimmunogen. For example, immunogens may be selected from a variety ofviral families. Example of desirable viral families against which animmune response would be desirable include, the picornavirus family,which includes the genera rhinoviruses, which are responsible for about50% of cases of the common cold; the genera enteroviruses, which includepolioviruses, coxsackieviruses, echoviruses, and human enterovirusessuch as hepatitis A virus; and the genera apthoviruses, which areresponsible for foot and mouth diseases, primarily in non-human animals.Within the picornavirus family of viruses, target antigens include theVP1, VP2, VP3, VP4, and VPG. Another viral family includes thecalcivirus family, which encompasses the Norwalk group of viruses, whichare an important causative agent of epidemic gastroenteritis. Stillanother viral family desirable for use in targeting antigens forinducing immune responses in humans and non-human animals is thetogavirus family, which includes the genera alphavirus, which includeSindbis viruses, RossRiver virus, and Venezuelan, Eastern & WesternEquine encephalitis, and rubivirus, including Rubella virus. Theflaviviridae family includes dengue, yellow fever, Japaneseencephalitis, St. Louis encephalitis and tick borne encephalitisviruses. Other target antigens may be generated from the Hepatitis C orthe coronavirus family, which includes a number of non-human virusessuch as infectious bronchitis virus (poultry), porcine transmissiblegastroenteric virus (pig), porcine hemagglutinating encephalomyelitisvirus (pig), feline infectious peritonitis virus (cats), feline entericcoronavirus (cat), canine coronavirus (dog), and human respiratorycoronaviruses, which may cause the common cold and/or non-A, B or Chepatitis. Within the coronavirus family, target antigens include the E1(also called M or matrix protein), E2 (also called S or Spike protein),E3 (also called HE or hemagglutin-elterose) glycoprotein (not present inall coronaviruses), or N (nucleocapsid). Still other antigens may betargeted against the rhabdovirus family, which includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus,parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. The reovirus family includes the genera reovirus,rotavirus (which causes acute gastroenteritis in children), orbiviruses,and cultivirus (Colorado Tick fever, Lebombo (humans), equineencephalosis, blue tongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Between the HIV and SIV, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat and Rev proteins, as well as various fragments thereof. In addition,a variety of modifications to these antigens have been described.Suitable antigens for this purpose are known to those of skill in theart. For example, one may select a sequence encoding the gag, pol, Vif,and Vpr, Env, Tat and Rev, amongst other proteins. See, e.g., themodified gag protein which is described in U.S. Pat. No. 5,972,596. See,also, the HIV and SIV proteins described in D. H. Barouch et al, J.Virol., 75(5):2462-2467 (March 2001), and R. R. Amara, et al, Science,292:69-74 (6 Apr. 2001). These proteins or subunits thereof may bedelivered alone, or in combination via separate vectors or from a singlevector.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O. B.) which cause respiratory disease and/orenteritis. The parvovirus family feline parvovirus (feline enteritis),feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (HCMV, muromegalovirus) andthe sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis,Marek=s disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and thesub-family entomopoxvirinae. The hepadnavirus family includes theHepatitis B virus. One unclassified virus which may be suitable sourceof antigens is the Hepatitis delta virus. Still other viral sources mayinclude avian infectious bursal disease virus and porcine respiratoryand reproductive syndrome virus. The alphavirus family includes equinearteritis virus and various Encephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci; andstreptococci. Pathogenic gram-negative cocci include meningococcus;gonococcus. Pathogenic enteric gram-negative bacilli includeenterobacteriaceae; pseudomonas, acinetobacteria and eikenella;melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi(which causes chancroid); brucella; Franisella tularensis (which causestularemia); yersinia (pasteurella); streptobacillus moniliformis andspirillum; Gram-positive bacilli include listeria monocytogenes;erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria);cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); andbartonellosis. Diseases caused by pathogenic anaerobic bacteria includetetanus; botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoans and helminths and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracia (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fever, all ofwhich are currently classified as

Category A agents; Coxiella burnetti (Q fever); Brucella species(brucellosis), Burkholderia mallei (glanders), Ricinus communis and itstoxin (ricin toxin), Clostridium perfringens and its toxin (epsilontoxin), Staphylococcus species and their toxins (enterotoxin B), all ofwhich are currently classified as Category B agents; and Nipan virus andhantaviruses, which are currently classified as Category C agents. Inaddition, other organisms, which are so classified or differentlyclassified, may be identified and/or used for such a purpose in thefuture. It will be readily understood that the viral vectors and otherconstructs described herein are useful to deliver antigens from theseorganisms, viruses, their toxins or other by-products, which willprevent and/or treat infection or other adverse reactions with thesebiological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding CTLs to eliminate those T cells. In rheumatoid arthritis (RA),several specific variable regions of T cell receptors (TCRs) which areinvolved in the disease have been characterized. These TCRs include V-3,V-14, V-17 and Vα-17. Thus, delivery of a nucleic acid sequence thatencodes at least one of these polypeptides will elicit an immuneresponse that will target T cells involved in RA. In multiple sclerosis(MS), several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include V-7 and Vα-10.Thus, delivery of a nucleic acid sequence that encodes at least one ofthese polypeptides will elicit an immune response that will target Tcells involved in MS. In scleroderma, several specific variable regionsof TCRs which are involved in the disease have been characterized. TheseTCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16,Vα-28 and Vα-12. Thus, delivery of a nucleic acid molecule that encodesat least one of these polypeptides will elicit an immune response thatwill target T cells involved in scleroderma.

Optionally, vectors containing AAV sequences of the invention may bedelivered using a prime-boost regimen. A variety of such regimens havebeen described in the art and may be readily selected. See, e.g., WO00/11140, published Mar. 2, 2000, incorporated by reference.

Such prime-boost regimens typically involve the administration of a DNA(e.g., plasmid) based vector to prime the immune system to second,booster, administration with a traditional antigen, such as a protein ora recombinant virus carrying the sequences encoding such an antigen. Inone embodiment, the invention provides a method of priming and boostingan immune response to a selected antigen by delivering a plasmid DNAvector carrying said antigen, followed by boosting, e.g., with a vectorcontaining AAV sequences of the invention.

In one embodiment, the prime-boost regimen involves the expression ofmultiproteins from the prime and/or the boost vehicle. See, e.g., R. R.Amara, Science, 292:69-74 (6 Apr. 2001) which describes a multiproteinregimen for expression of protein subunits useful for generating animmune response against HIV and SIV. For example, a DNA prime maydeliver the Gag, Pol, Vif, VPX and Vpr and Env, Tat, and Rev from asingle transcript. Alternatively, the SIV Gag, Pol and HIV-1 Env isdelivered.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first chimp vector of the invention followed byboosting with a second chimp vector, or with a composition containingthe antigen itself in protein form. In one or example, the prime-boostregimen can provide a protective immune response to the virus, bacteriaor other organism from which the antigen is derived. In another desiredembodiment, the prime-boost regimen provides a therapeutic effect thatcan be measured using convention assays for detection of the presence ofthe condition for which therapy is being administered.

The priming vaccine may be administered at various sites in the body ina dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The invention is not limitedto the amount or situs of injection(s) or to the pharmaceutical carrier.Rather, the priming step encompasses treatment regimens which include asingle dose or dosage which is administered hourly, daily, weekly ormonthly, or yearly. As an example, the mammals may receive one or twopriming injection containing between about 10 μg to about 50 μg ofplasmid in carrier. A desirable priming amount or dosage of the primingDNA vaccine composition ranges between about 1 μg to about 10,000 μg ofthe DNA vaccine. Dosages may vary from about 1 μg to 1000 μg DNA per kgof subject body weight. The amount or site of injection is desirablyselected based upon the identity and condition of the mammal beingvaccinated.

The dosage unit of the DNA vaccine suitable for delivery of the antigento the mammal is described herein. The DNA vaccine is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline, isotonicsalts solution or other formulations which will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions of the invention may be administeredto a mammal according to the routes described above, in a sustainedrelease formulation using a biodegradable biocompatible polymer, or byon-site delivery using micelles, gels and liposomes.

Optionally, the priming step of this invention also includesadministering with the priming DNA vaccine composition, a suitableamount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming DNA vaccine to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting vaccine composition containingor capable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source or from another source.Alternatively, the “boosting composition” can be a compositioncontaining the same antigen as encoded in the priming DNA vaccine, butin the form of a protein or peptide, which composition induces an immuneresponse in the host. In another embodiment, the boosting vaccinecomposition includes a composition containing a DNA sequence encodingthe antigen under the control of a regulatory sequence directing itsexpression in a mammalian cell, e.g., vectors such as well-knownbacterial or viral vectors. The primary requirements of the boostingvaccine composition are that the antigen of the vaccine composition isthe same antigen, or a cross-reactive antigen, as that encoded by theDNA vaccine.

Suitably, the vectors of the invention are also well suited for use inregimens which use non-AAV vectors as well as proteins, peptides, and/orother biologically useful therapeutic or immunogenic compounds. Theseregimens are particularly well suited to gene delivery for therapeuticposes and for immunization, including inducing protective immunity. Suchuses will be readily apparent to one of skill in the art.

Further, a vector of the invention provides an efficient gene transfervehicle which can deliver a selected transgene to a selected host cellin vivo or ex vivo even where the organism has neutralizing antibodiesto one or more AAV serotypes. In one embodiment, the vector (e.g., anrAAV) and the cells are mixed ex vivo; the infected cells are culturedusing conventional methodologies; and the transduced cells arere-infused into the patient. Further, the vectors of the invention mayalso be used for production of a desired gene product in vitro. For invitro production, a desired product (e.g., a protein) may be obtainedfrom a desired culture following transfection of host cells with a rAAVcontaining the molecule encoding the desired product and culturing thecell culture under conditions which permit expression. The expressedproduct may then be purified and isolated, as desired. Suitabletechniques for transfection, cell culturing, purification, and isolationare known to those of skill in the art.

The following examples illustrate several aspects and embodiments of theinvention.

EXAMPLES Example 1 PCR Amplification, Cloning and Characterization ofNovel AAV Sequences

Tissues from nonhuman primates were screened for AAV sequences using aPCR method based on oligonucleotides to highly conserved regions ofknown AAVs. A stretch of AAV sequence spanning 2886 to 3143 bp of AAV1[SEQ ID NO:61 was selected as a PCR amplicon in which a hypervariableregion of the capsid protein (Cap) that is unique to each known AAVserotype, which is termed herein a “signature region,” is flanked byconserved sequences. In later analysis, this signature region was shownto be located between conserved residues spanning hypervariable region3.

An initial survey of peripheral blood of a number of nonhuman primatespecies revealed detectable AAV in a subset of animals from species suchas rhesus macaques, cynomologous macaques, chimpanzees and baboons.However, there were no AAV sequences detected in some other speciestested, including Japanese macaques, pig-tailed macaques and squirrelmonkeys. A more extensive analysis of vector distribution was conductedin tissues of rhesus monkeys of the University of Pennsylvania andTulane colonies recovered at necropsy. This revealed AAV sequencethroughout a wide array of tissues.

A. Amplification of an AAV Signature Region

DNA sequences of AAV1-6 and AAVs isolated from Goose and Duck werealigned to each other using “Clustal W” at default settings. Thealignment for AAV1-6, and including the information for the novel AAV7,is provided in FIG. 1. Sequence similarities among AAVs were compared.

In the line of study, a 257 bp region spanning 2886 bp to 3143 bp of AAV1 [SEQ ID NO: 6], and the corresponding region in the genomes of AAV 2-6genomes [See, FIG. 1], was identified by the inventors. This region islocated with the AAV capsid gene and has highly conserved sequencesamong at both 5′ and 3′ ends and is relatively variable sequence in themiddle. In addition, this region contains a DraIII restriction enzymesite (CACCACGTC, SEQ ID NO:15). The inventors have found that thisregion serves as specific signature for each known type of AAV DNA. Inother words, following PCR reactions, digestion with endonucleases thatare specific to each known serotypes and gel electrophoresis analysis,this regions can be used to definitively identify amplified DNA as beingfrom serotype 1, 2, 3, 4, 5, 6, or another serotype.

The primers were designed, validated and PCR conditions optimized withAAV1, 2 and 5 DNA controls. The primers were based upon the sequences ofAAV2: 5′ primer, 1S: bp 2867-2891 of AAV2 (SEQ ID NO:7) and 3′ primer,18as, bp 3095-3121 of AAV2 (SEQ ID NO:7).

Cellular DNAs from different tissues including blood, brain, liver,lung, testis, etc. of different rhesus monkeys were studied utilizingthe strategy described above. The results revealed that DNAs fromdifferent tissues of these monkeys gave rise to strong PCRamplifications. Further restriction analyses of PCR products indicatedthat they were amplified from AAV sequences different from any publishedAAV sequences.

PCR products (about 255 bp in size) from DNAs of a variety of monkeytissues have been cloned and sequenced. Bioinformatics study of thesenovel AAV sequences indicated that they are novel AAV sequences ofcapsid gene and distinct from each other. FIG. 1 includes in thealignment the novel AAV signature regions for AAV10-12 [SEQ ID NO:117,118 and 119, respectively]. Multiple sequence alignment analysis wasperformed using the Clustal W (1.81) program. The percentage of sequenceidentity between the signature regions of AAV 1-7 and AAV 10-12 genomesis provided below.

TABLE 2 Sequences for Analysis Sequence # AAV Serotype Size (bp) 1 AAV1258 2 AAV2 255 3 AAV3 255 4 AAV4 246 5 AAV5 258 6 AAV6 258 7 AAV7 258 10AAV10 255 11 AAV11 258 12 AAV12 255

TABLE 3 Pairwise Alignment (Percentage of Identity) AAV2 AAV3 AAV4 AAV5AAV6 AAV7 AAV10 AAV11 AAV12 AAV1 90 90 81 76 97 91 93 94 93 AAV2 93 7978 90 90 93 93 92 AAV3 80 76 90 92 92 92 92 AAV4 76 81 84 82 81 79 AAV575 78 79 79 76 AAV6 91 92 94 94 AAV7 94 92 92 AAV10 95 93 AAV11 94

Over 300 clones containing novel AAV serotype sequences that span theselected 257 bp region were isolated and sequenced. Bioinformaticsanalysis of these 300+ clones suggests that this 257 bp region iscritical in serving as a good land marker or signature sequence forquick isolation and identification of novel AAV serotype.

B. Use of the Signature Region for PCR Amplification.

The 257 bp signature region was used as a PCR anchor to extend PCRamplifications to 5′ of the genome to cover the junction region of repand cap genes (1398 bp-3143 bp, SEQ ID NO:6) and 3′ of the genome toobtain the entire cap gene sequence (2866 bp-4600 bp, SEQ ID NO:6). PCRamplifications were carried out using the standard conditions, includingdenaturing at 95° C. for 0.5-1 min, annealing at 60-65° C. for 0.5-1 minand extension at 72° C. for 1 min per kb with a total number ofamplification cycles ranging from 28 to 42.

Using the aligned sequences as described in “A”, two other relativeconserved regions were identified in the sequence located in 3′ end ofrep genes and 5′ to the 257 bp region and in the sequence down stream ofthe 257 bp fragment but before the AAV′ 3 ITR. Two sets of new primerswere designed and PCR conditions optimized for recovery of entire capsidand a part of rep sequences of novel AAV serotypes. More specifically,for the 5′ amplification, the 5′ primer, AV1Ns, wasGCTGCGTCAACTGGACCAATGAGAAC [nt 1398-1423 of AAV1, SEQ ID NO:6] and the3′ primer was 18as, identified above. For the 3′ amplification, the 5′primer was 1 s, identified above, and the 3′ primer was AV2Las,TCGTTTCAGTTGAACTTTGGTCTCTGCG [nt 4435-4462 of AAV2, SEQ ID NO:7].

In these PCR amplifications, the 257 bp region was used as a PCR anchorand land marker to generate overlapping fragments to construct acomplete capsid gene by fusion at the DraIII site in the signatureregion following amplification of the 5′ and 3′ extension fragmentsobtained as described herein. More particularly, to generate the intactAAV7 cap gene, the three amplification products (a) the sequences of thesignature region; (b) the sequences of the 5′ extension; and (c) thesequences of the 3′ extension were cloned into a pCR4-Topo [Invitrogen]plasmid backbone according to manufacturer's instructions. Thereafter,the plasmids were digested with DraIII and recombined to form an intactcap gene.

In this line of work, about 80% of capsid sequences of AAV7 and AAV 8were isolated and analyzed. Another novel serotype, AAV9, was alsodiscovered from Monkey #2.

Using the PCR conditions described above, the remaining portion of therep gene sequence for AAV7 is isolated and cloned using the primers thatamplify 108 bp to 1461 bp of AAV genome (calculated based on thenumbering of AAV2, SEQ ID NO:7). This clone is sequenced forconstruction of a complete AAV7 genome without ITRs.

C. Direct Amplification of 3.1 kb Cap Fragment

To directly amplify a 3.1 kb full-length Cap fragment from NHP tissueand blood DNAs, two other highly conserved regions were identified inAAV genomes for use in PCR amplification of large fragments. A primerwithin a conserved region located in the middle of the rep gene wasselected (AV1ns: 5′ GCTGCGTCAACTGGACCAATGAGAAC 3′, nt 1398-1423 of SEQID NO:6) in combination with the 3′ primer located in another conservedregion downstream of the Cap gene (AV2cas: 5′CGCAGAGACCAAAGTTCAACTGAAACGA 3′, SEQ ID NO:7) for amplification offull-length cap fragments. The PCR products were Topo-cloned accordingto manufacturer's directions (Invitrogen) and sequence analysis wasperformed by Qiagengenomics (Qiagengenomics, Seattle, Wash.) with anaccuracy of 99.9%. A total of 50 capsid clones were isolated andcharacterized. Among them, 37 clones were derived from Rhesus macaquetissues (rh.1-rh.37), 6 clones from cynomologous macaques (cy.1-cy.6), 2clones from Baboons (bb.1 and bb.2) and 5 clones from Chimps(ch.1-ch.5).

To rule out the possibility that sequence diversity within the novel AAVfamily was not an artifact of the PCR, such as PCR-mediated genesplicing by overlap extension between different partial DNA templateswith homologous sequences, or the result of recombination process inbacteria, a series of experiments were performed under identicalconditions for VP1 amplification using total cellular DNAs. First,intact AAV7 and AAV8 plasmids were mixed at an equal molar ratiofollowed by serial dilutions. The serially diluted mixtures were used astemplates for PCR amplification of 3.1 kb VP1 fragments using universalprimers and identical PCR conditions to that were used for DNAamplifications to see whether any hybrid PCR products were generated.The mixture was transformed into bacteria and isolated transformants tolook for hybrid clones possibly derived from recombination process inbacterial cells. In a different experiment, we restricted AAV7 and AAV8plasmids with Msp I, Ava I and HaeI, all of which cut both genomesmultiple times at different positions, mixed the digestions in differentcombinations and used them for PCR amplification of VP1 fragments underthe same conditions to test whether any PCR products could be generatedthrough overlap sequence extension of partial AAV sequences. In anotherexperiment, a mixture of gel purified 5′ 1.5 kb AAV7 VP1 fragment and 3′1.7 kb AAV8 VP1 fragment with overlap in the signature region wasserially diluted and used for PCR amplification in the presence andabsence of 200 ng cellular DNA extracted from a monkey cell line thatwas free of AAV sequences by TaqMan analysis. None of these experimentsdemonstrated efficient PCR-mediated overlap sequence production underthe conditions of the genomic DNA Cap amplification (data not shown). Asa further confirmation, 3 pairs of primers were designed, which werelocated at different HVRs, and were sequence specific to the variants ofclone 42 s from Rhesus macaque F953, in different combinations toamplify shorter fragments from mesenteric lymph node (MLN) DNA from F953from which clone 42 s were isolated. All sequence variations identifiedin full-length Cap clones were found in these short fragments (data notshown).

Example 2 Adeno-Associated Viruses Undergo Substantial Evolution inPrimates During Natural Infections

Sequence analysis of selected AAV isolates revealed divergencethroughout the genome that is most concentrated in hypervariable regionsof the capsid proteins. Epidemiologic data indicate that all knownserotypes are endemic to primates, although isolation of clinicalisolates has been restricted to AAV2 and AAV3 from anal and throat swabsof human infants and AAV5 from a human condylomatous wart. No knownclinical sequalae have been associated with AAV infection.

In an attempt to better understand the biology of AAV, nonhuman primateswere used as models to characterize the sequlae of natural infections.Tissues from nonhuman primates were screened for AAV sequences using thePCR method of the invention based on oligonucleotides to highlyconserved regions of known AAVs (see Example 1). A stretch of AAVsequence spanning 2886 to 3143 bp of AAV1 [SEQ ID NO:61 was selected asa PCR amplicon in which conserved sequences are flanked by ahypervariable region that is unique to each known AAV serotype, termedherein a “signature region.”

An initial survey of peripheral blood of a number of nonhuman primatespecies including rhesus monkeys, cynomologous monkeys, chimpanzees, andbaboons revealed detectable AAV in a subset of animals from all species.A more extensive analysis of vector distribution was conducted intissues of rhesus monkeys of the University of Pennsylvania and Tulanecolonies recovered at necropsy. This revealed AAV sequence throughout awide array of tissues.

The amplified signature sequences were subcloned into plasmids andindividual transformants were subjected to sequence analysis. Thisrevealed substantial variation in nucleotide sequence of clones derivedfrom different animals. Variation in the signature sequence was alsonoted in clones obtained within individual animals. Tissues harvestedfrom two animals in which unique signature sequences were identified(i.e., colon from 98E044 and heart from 98E056) were furthercharacterized by expanding the sequence amplified by PCR usingoligonucleotides to highly conserved sequences. In this way, completeproviral structures were reconstructed for viral genomes from bothtissues as described herein. These proviruses differ from the otherknown AAVs with the greatest sequence divergence noted in regions of theCap gene.

Additional experiments were performed to confirm that AAV sequencesresident to the nonhuman primate tissue represented proviral genomes ofinfectious virus that is capable of being rescued and form virions.Genomic DNA from liver tissue of animal 98E056, from which AAV8signature sequence was detected, was digested with an endonuclease thatdoes not have a site within the AAV sequence and transfected into 293cells with a plasmid containing an E 1 deleted genome of humanadenovirus serotype 5 as a source of helper functions. The resultinglysate was passaged on 293 cells once and the lysate was recovered andanalyzed for the presence of AAV Cap proteins using a broadly reactingpolyclonal antibody to Cap proteins and for the presence and abundanceof DNA sequences from the PCR amplified AAV provirus from which AAV8 wasderived. Transfection of endonuclease restricted heart DNA and theadenovirus helper plasmid yielded high quantities of AAV8 virus asdemonstrated by the detection of Cap proteins by Western blot analysisand the presence of 10⁴ AAV8 vector genomes per 293 cell. Lysates weregenerated from a large-scale preparation and the AAV was purified bycesium sedimentation. The purified preparation demonstrated 26 nmicosohedral structures that look identical to those of AAV serotype 2.Transfection with the adenovirus helper alone did not yield AAV proteinsor genomes, ruling out contamination as a source of the rescued AAV.

To further characterize the inter and intra animal variation of AAVsignature sequence, selected tissues were subjected to extended PCR toamplify entire Cap open reading frames.

The resulting fragments were cloned into bacterial plasmids andindividual transformants were isolated and fully sequenced. Thisanalysis involved mesenteric lymph nodes from three rhesus monkeys(Tulane/V223—6 clones; Tulane/T612—7 clones; Tulane/F953—14 clones),liver from two rhesus monkeys (TulaneN251—3 clones; Penn/00E033—3clones), spleen from one rhesus monkey (Penn/97E043—3 clones), heartfrom one rhesus monkey (IHGT/98E046—1 clone) and peripheral blood fromone chimpanzee (New Iberia/X133—5 clones), six cynomologous macaques(Charles River/A1378, A3099, A3388, A3442, A2821, A3242—6 clones total)and one Baboon (SFRB/8644—2 clones). Of the 50 clones that weresequenced from 15 different animals, 30 were considered non-redundantbased on the finding of at least 7 amino acid differences from oneanother. The non-redundant VP1 clones are numbered sequentially as theywere isolated, with a prefix indicating the species of non-human primatefrom which they were derived. The structural relationships between these30 non-redundant clones and the previously described 8 AAV serotypeswere determined using the SplitsTree program [Huson, D. H. SplitsTree:analyzing and visualizing evolutionary data. Bioinformatics 14, 68-73(1998)] with implementation of the method of split decomposition. Theanalysis depicts homoplasy between a set of sequences in a tree-likenetwork rather than a bifurcating tree. The advantage is to enabledetection of groupings that are the result of convergence and to exhibitphylogenetic relationships even when they are distorted by parallelevents. Extensive phylogenetic research will be required in order toelucidate the AAV evolution, whereas the intention here only is to groupthe different clones as to their sequence similarity.

To confirm that the novel VP1 sequences were derived from infectiousviral genomes, cellular DNA from tissues with high abundance of viralDNA was restricted with an endonuclease that should not cleave withinAAV and transfected into 293 cells, followed by infection withadenovirus. This resulted in rescue and amplification of AAV genomesfrom DNA of tissues from two different animals (data not shown).

VP1 sequences of the novel AAVs were further characterized with respectto the nature and location of amino acid sequence variation. All 30 VP1clones that were shown to differ from one another by greater than 1%amino acid sequence were aligned and scored for variation at eachresidue. An algorithm developed to determine areas of sequencedivergence yielded 12 hypervariable regions (HVR) of which 5 overlap orare part of the 4 previously described variable regions [Kotin, citedabove; Rutledge, cited above]. The three-fold-proximal peaks containmost of the variability (HVR5-10). Interestingly the loops located atthe 2 and 5 fold axis show intense variation as well. The HVRs 1 and 2occur in the N-terminal portion of the capsid protein that is notresolved in the X-ray structure suggesting that the N-terminus of theVP1 protein is exposed on the surface of the virion.

Real-time PCR was used to quantify AAV sequences from tissues of 21rhesus monkeys using primers and probes to highly conserved regions ofRep (one set) and Cap (two sets) of known AAVs. Each data pointrepresents analysis from tissue DNA from an individual animal Thisconfirmed the wide distribution of AAV sequences, although thequantitative distribution differed between individual animals. Thesource of animals and previous history or treatments did not appear toinfluence distribution of AAV sequences in rhesus macaques. The threedifferent sets of primers and probes used to quantify AAV yieldedconsistent results. The highest levels of AAV were found consistently inmesenteric lymph nodes at an average of 0.01 copies per diploid genomefor 13 animals that were positive. Liver and spleen also contained highabundance of virus DNA. There were examples of very high AAV, such as inheart of rhesus macaque 98E056, spleen of rhesus macaque 97E043 andliver of rhesus macaque RQ4407, which demonstrated 1.5, 3 and 20 copiesof AAV sequence per diploid genome respectively. Relatively low levelsof virus DNA were noted in peripheral blood mononuclear cells,suggesting the data in tissue are not due to resident blood components(data not shown). It should be noted that this method would notnecessarily capture all AAVs resident to the nonhuman primates sincedetection requires high homology to both the oligonucleotides and thereal time PCR probe. Tissues from animals with high abundance AAV DNAwas further analyzed for the molecular state of the DNA, by DNAhybridization techniques, and its cellular distribution, by in situhybridization.

The kind of sequence variation revealed in AAV proviral fragmentsisolated from different animals and within tissues of the same animalsis reminiscent of the evolution that occurs for many RNA viruses duringpandemics or even within the infection of an individual. In somesituations the notion of a wild-type virus has been replaced by theexistence of swarms of quasispecies that evolve as a result of rapidreplication and mutations in the presence of selective pressure. Oneexample is infection by HIV, which evolves in response to immunologicand pharmacologic pressure. Several mechanisms contribute to the highrate of mutations in RNA viruses, including low fidelity and lack ofproof reading capacity of reverse transcriptase and non-homologous andhomologous recombination.

Evidence for the formation of quasispecies of AAV was illustrated inthis study by the systematic sequencing of multiple cloned proviralfragments. In fact, identical sequences could not be found within anyextended clones isolated between or within animals. An importantmechanism for this evolution of sequence appears to be a high rate ofhomologous recombination between a more limited number of parenteralviruses. The net result is extensive swapping of hypervariable regionsof the Cap protein leading to an array of chimeras that could havedifferent tropisms and serologic specificities (i.e., the ability toescape immunologic responses especially as it relates to neutralizingantibodies). Mechanisms by which homologous recombination could occurare unclear. One possibility is that + and − strands of different singlestranded AAV genomes anneal during replication as has been describedduring high multiplicity of infections with AAV recombinants. It isunclear if other mechanisms contribute to sequence evolution in AAVinfections. The overall rate of mutation that occurs during AAVreplication appears to be relatively low and the data do not suggesthigh frequencies of replication errors. However, substantialrearrangements of the AAV genome have been described during lyticinfection leading to the formation of defective interfering particles.Irrespective of the mechanisms that lead to sequence divergence, withfew exceptions, vp1 structures of the quasispecies remained intactwithout frameshifts or nonsense mutations suggesting that competitiveselection of viruses with the most favorable profile of fitnesscontribute to the population dynamics.

These studies have implications in several areas of biology andmedicine. The concept of rapid virus evolution, formerly thought to be aproperty restricted to RNA viruses, should be considered in DNA viruses,which classically have been characterized by serologic assays. It willbe important in terms of parvoviruses to develop a new method fordescribing virus isolates that captures the complexity of its structureand biology, such as with HIV, which are categorized as general familiesof similar structure and function called Clades. An alternative strategyis to continue to categorize isolates with respect to serologicspecificity and develop criteria for describing variants withinserologic groups.

Example 3 Vectorology of Recombinant AAV Genomes Equipped with AAV2 ITRsis Using Chimeric Plasmids Containing AAV2 Rep and Novel AAV Cap Genesfor Serological and Gene Transfer Studies in Different Animal Models

Chimeric packaging constructs are generated by fusing AAV2 rep with capsequences of novel AAV serotypes. These chimeric packaging constructsare used, initially, for pseudotyping recombinant AAV genomes carryingAAV2 ITRs by triple transfection in 293 cell using Ad5 helper plasmid.These pseudotyped vectors are used to evaluate performance intransduction-based serological studies and evaluate gene transferefficiency of novel AAV serotypes in different animal models includingNHP and rodents, before intact and infectious viruses of these novelserotypes are isolated.

A. pAAV2GFP

The AAV2 plasmid which contains the AAV2 ITRs and green fluorescentprotein expressed under the control of a constitutitive promoter. Thisplasmid contains the following elements: the AAV2 ITRs, a CMV promoter,and the GFP coding sequences.

B. Cloning of Trans Plasmid

To construct the chimeric trans-plasmid for production of recombinantpseudotyped AAV7 vectors, p5E18 plasmid (Xiao et al., 1999, J. Virol73:3994-4003) was partially digested with Xho I to linearize the plasmidat the Xho I site at the position of 3169 bp only. The Xho I cut endswere then filled in and ligated back. This modified p5E18 plasmid wasrestricted with Xba I and Xho I in a complete digestion to remove theAAV2 cap gene sequence and replaced with a 2267 bp Spe I/Xho I fragmentcontaining the AAV7 cap gene which was isolated from pCRAAV7 6-5+15-4plasmid.

The resulting plasmid contains the AAV2 rep sequences for Rep78/68 underthe control of the AAV2 P5 promoter, and the AAV2 rep sequences forRep52/40 under the control of the AAV2 P19 promoter. The AAV7 capsidsequences are under the control of the AAV2 P40 promoter, which islocated within the Rep sequences. This plasmid further contains a spacer5′ of the rep ORF.

C. Production of Pseudotyped rAAV

The rAAV particles (AAV2 vector in AAV7 capsid) are generated using anadenovirus-free method. Briefly, the cis plasmid (pAAV2.1 lacZ plasmidcontaining AAV2 ITRs), and the trans plasmid pCRAAV7 6-5+15-4(containing the AAV2 rep and AAV7 cap) and a helper plasmid,respectively, were simultaneously co-transfected into 293 cells in aratio of 1:1:2 by calcium phosphate precipitation.

For the construction of the pAd helper plasmids, pBG10 plasmid waspurchased from Microbix (Canada). A RsrII fragment containing L2 and L3was deleted from pBHG10, resulting in the first helper plasmid, pAdαF13.Plasmid AdΔ F1 was constructed by cloning Asp700/SalI fragment with aPmeI/Sgfl deletion, isolating from pBHG10, into Bluescript. MLP, L2, L2and L3 were deleted in the pAdΔF1. Further deletions of a 2.3 kb NruIfragment and, subsequently, a 0.5 kb RsrII/NruI fragment generatedhelper plasmids pAdΔF5 and pAdΔF6, respectively. The helper plasmid,termed pΔF6, provides the essential helper functions of E2a and E4 ORF6not provided by the E1-expressing helper cell, but is deleted ofadenoviral capsid proteins and functional E1 regions).

Typically, 50 μg of DNA (cis:trans:helper) was transfected onto a 150 mmtissue culture dish. The 293 cells were harvested 72 hourspost-transfection, sonicated and treated with 0.5% sodium deoxycholate(37EC for 10 min) Cell lysates were then subjected to two rounds of aCsCl gradient. Peak fractions containing rAAV vector are collected,pooled and dialyzed against PBS.

Example 4 Creation of Infectious Clones Carrying Intact Novel AAVSerotypes for Study of Basic Virology in Human and NHP Derived CellLines and Evaluation of Pathogenesis of Novel AAV Serotypes in NHP andOther Animal Models

To achieve this goal, the genome walker system is employed to obtain 5′and 3′ terminal sequences (ITRs) and complete construction of clonescontaining intact novel AAV serotype genomes.

Briefly, utilizing a commercially available Universal Genome Walker Kit[Clontech], genomic DNAs from monkey tissues or cell lines that areidentified as positive for the presence of AAV7 sequence are digestedwith Dra I, EcoR V, Pvu II and Stu I endonucleases and ligated to GenomeWalker Adaptor to generate 4 individual Genome Walker Libraries (GWLs).Using DNAs from GWLs as templates, AAV7 and adjacent genomic sequenceswill be PCR-amplified by the adaptor primer 1 (AP1, provided in the kit)and an AAV7 specific primer 1, followed by a nested PCR using theadaptor primer 2 (AP2) and another AAV7 specific primer 2, both of whichare internal to the first set of primers. The major PCR products fromthe nested PCR are cloned and characterized by sequencing analysis.

In this experiment, the primers covering the 257 bp or other signaturefragment of a generic AAV genome are used for PCR amplification ofcellular DNAs extracted from Human and NHP derived cell lines toidentify and characterize latent AAV sequences. The identified latentAAV genomes are rescued from the positive cell lines using adenovirushelpers of different species and strains.

To isolate infectious AAV clones from NHP derived cell lines, a desiredcell line is obtained from ATCC and screened by PCR to identify the 257bp amplicon, i.e., signature region of the invention. The 257 bp PCRproduct is cloned and serotyped by sequencing analysis. For these celllines containing the AAV7 sequence, the cells are infected with SV-15, asimian adenovirus purchased from ATCC, human Ad5 or transfected withplasmid construct housing the human Ad genes that are responsible forAAV helper functions. At 48 hour post infection or transfection, thecells are harvested and Hirt DNA is prepared for cloning of AAV7 genomefollowing Xiao et al., 1999, J. Virol, 73:3994-4003.

Example 5 Production of AAV Vectors

A pseudotyping strategy similar to that of Example 3 for AAV1/7 wasemployed to produce AAV2 vectors packaged with AAV1, AAV5 and AAV8capsid proteins. Briefly, recombinant AAV genomes equipped with AAV2ITRs were packaged by triple transfection of 293 cells with cis-plasmid,adenovirus helper plasmid and a chimeric packaging construct where theAAV2 rep gene is fused with cap genes of novel AAV serotypes. To createthe chimeric packaging constructs, the Xho I site of p5E18 plasmid at3169 bp was ablated and the modified plasmid was restricted with Xba Iand Xho I in a complete digestion to remove the AAV2 cap gene andreplace it with a 2267 bp Spe I/Xho I fragment containing the AAV8 capgene [Xiao, W., et al., (1999) J Virol 73, 3994-4003]. A similar cloningstrategy was used for creation of chimeric packaging plasmids of AAV2/1and AAV2/5. All recombinant vectors were purified by the standard CsCl₂sedimentation method except for AAV2/2, which was purified by singlestep heparin chromatography.

Genome copy (GC) titers of AAV vectors were determined by TaqMananalysis using probes and primers targeting SV40 poly A region asdescribed previously [Gao, G., et al., (2000) Hum Gene Ther 11,2079-91].

Vectors were constructed for each serotype for a number of in vitro andin vivo studies. Eight different transgene cassettes were incorporatedinto the vectors and recombinant virions were produced for eachserotype. The recovery of virus, based on genome copies, is summarizedin Table 4 below. The yields of vector were high for each serotype withno consistent differences between serotypes. Data presented in the tableare average genome copy yields with standard deviation×10¹³ of multipleproduction lots of 50 plate (150 mm) transfections.

TABLE 4 Production of Recombinant Vectors AAV2/1 AAV2/2 AAV2/5 AAV2/7AAV2/8 CMV 7.30 ± 4.33 4.49 ± 2.89 5.19 ± 5.19 3.42 0.87 LacZ (n = 9) (n= 6) (n = 8) (n = 1) (n = 1) CMV 6.43 ± 2.42 3.39 ± 2.42 5.55 ± 6.492.98 ± 2.66 3.74 ± 3.88 EGFP (n = 2) (n = 2) (n = 4) (n = 2) (n = 2) TBGLacZ 4.18  0.23 0.704 ± 0.43  2.16  0.532 (n = 1) (n = 1) (n = 2) (n= 1) (n = 1) Alb A1AT 4.67 ± 0.75 4.77 4.09 5.04 2.02 (n = 2) (n = 1) (n= 1) (n = 1) (n = 1) CB A1AT 0.567  0.438 2.82 2.78 0.816 ± 0.679 (n= 1) (n = 1) (n = 1) (n = 1) (n = 2) TBG 8.51 ± 6.65 3.47 ± 2.09 5.26 ±3.85 6.52 ± 3.08 1.83 ± 0.98 rhCG (n = 6) (n = 5) (n = 4) (n = 4) (n =5) TBG cFIX 1.24 ± 1.29  0.63 ± 0.394 3.74 ± 2.48 4.05 15.8 ± 15.0 (n =3) (n = 6) (n = 7) (n = 1) (n = 5)

Example 6 Serologic Analysis of Pseudotyped Vectors

C57BL/6 mice were injected with vectors of different serotypes ofAAVCBA1AT vectors intramuscularly (5×10¹¹ GC) and serum samples werecollected 34 days later. To test neutralizing and cross-neutralizingactivity of sera to each serotype of AAV, sera was analyzed in atransduction based neutralizing antibody assay [Gao, G. P., et al.,(1996) J Virol 70, 8934-43]. More specifically, the presence ofneutralizing antibodies was determined by assessing the ability of serumto inhibit transduction of 84-31 cells by reporter viruses (AAVCMVEGFP)of different serotypes. Specifically, the reporter virus AAVCMVEGFP ofeach serotype [at multiplicity of infection (MOI) that led to atransduction of 90% of indicator cells] was pre-incubated withheat-inactivated serum from animals that received different serotypes ofAAV or from naïve mice. After 1-hour incubation at 37° C., viruses wereadded to 84-31 cells in 96 well plates for 48 or 72-hour, depending onthe virus serotype. Expression of GFP was measured by FluoroImagin(Molecular Dynamics) and quantified by Image Quant Software.Neutralizing antibody titers were reported as the highest serum dilutionthat inhibited transduction to less than 50%.

The availability of GFP expressing vectors simplified the development ofan assay for neutralizing antibodies that was based on inhibition oftransduction in a permissive cell line (i.e., 293 cells stablyexpressing E4 from Ad5). Sera to selected AAV serotypes were generatedby intramuscular injection of the recombinant viruses. Neutralization ofAAV transduction by 1:20 and 1:80 dilutions of the antisera wasevaluated (See Table 5 below). Antisera to AAV1, AAV2, AAV5 and AAV8neutralized transduction of the serotype to which the antiserum wasgenerated (AAV5 and AAV8 to a lesser extent than AAV1 and AAV2) but notto the other serotype (i.e., there was no evidence of crossneutralization suggesting that AAV 8 is a truly unique serotype).

TABLE 5 Serological Analysis of New AAV Serotypes. % Infection on 84-31cells with AAVCMVEGFP virus: AAV2/1 AAV2/2 AAV2/5 AAV2/7 AAV2/8 SerumSerum Serum Serum Serum Immunization dilution: dilution: dilution:dilution: dilution: Sera: Vector 1/20 1/80 1/20 1/80 1/20 1/80 1/20 1/801/20 1/80 Group 1 AAV2/1 0 0 100 100 100 100 100 100 100 100 Group 2AAV2/2 100 100 0 0 100 100 100 100 100 100 Group 3 AAV2/5 100 100 100100 16.5 16.5 100 100 100 100 Group 4 AAV2/7 100 100 100 100 100 10061.5 100 100 100 Group 5 AAV2/8 100 100 100 100 100 100 100 100 26.3 60

Human sera from 52 normal subjects were screened for neutralizationagainst selected serotypes. No serum sample was found to neutralizeAAV2/7 and AAV2/8 while AAV2/2 and AAV2/1 vectors were neutralized in20% and 10% of sera, respectively. A fraction of human pooled IgGrepresenting a collection of 60,000 individual samples did notneutralize AAV2/7 and AAV2/8, whereas AAV2/2 and AAV2/1 vectors wereneutralized at titers of serum equal to 1/1280 and 1/640, respectively.

Example 7 In vivo Evaluation of Different Serotypes of AAV Vectors

In this study, 7 recombinant AAV genomes, AAV2CBhA1AT, AAV2AlbhA1AT,AAV2CMVrhCG, AAV2TBGrhCG, AAV2TBGcFIX, AAV2CMVLacZ and AAV2TBGLacZ werepackaged with capsid proteins of different serotypes. In all 7constructs, minigene cassettes were flanked with AAV2 ITRs. cDNAs ofhuman α-antitrypsin (A1AT) [Xiao, W., et al., (1999) J Virol 73,3994-4003] β-subunit of rhesus monkey choriogonadotropic hormone (CG)[Zoltick, P. W. & Wilson, J. M. (2000) Mol Ther 2, 657-9] canine factorIX [Wang, L., et al., (1997) Proc Natl Acad Sci USA 94, 11563-6] andbacterial β-glactosidase (i.e., Lac Z) genes were used as reportergenes. For liver-directed gene transfer, either mouse albumin genepromoter (Alb) [Xiao, W. (1999), cited above] or human thyroid hormonebinding globulin gene promoter (TBG) [Wang (1997), cited above] was usedto drive liver specific expression of reporter genes. In muscle-directedgene transfer experiments, either cytomegalovirus early promoter (CMV)or chicken β-actin promoter with CMV enhancer (CB) was employed todirect expression of reporters.

For muscle-directed gene transfer, vectors were injected into the righttibialis anterior of 4-6 week old NCR nude or C57BL/6 mice (Taconic,Germantown, N.Y.). In liver-directed gene transfer studies, vectors wereinfused intraportally into 7-9 week old NCR nude or C57BL/6 mice(Taconic, Germantown, N.Y.). Serum samples were collected intraorbitallyat different time points after vector administration. Muscle and livertissues were harvested at different time points for cryosectioning andXgal histochemical staining from animals that received the lacZ vectors.For the re-administration experiment, C56BL/6 mice initially receivedAAV2/1, 2/2, 2/5, 2/7 and 2/8CBA1AT vectors intramuscularly and followedfor A1AT gene expression for 7 weeks. Animals were then treated withAAV2/8TBGcFIX intraportally and studied for cFIX gene expression.

ELISA based assays were performed to quantify serum levels of hA1AT,rhCG and cFIX proteins as described previously [Gao, G. P., et al.,(1996) J Virol 70, 8934-43; Zoltick, P. W. & Wilson, J. M. (2000) MolTher 2, 657-9; Wang, L., et al., Proc Natl Acad Sci USA 94, 11563-6].The experiments were completed when animals were sacrificed for harvestof muscle and liver tissues for DNA extraction and quantitative analysisof genome copies of vectors present in target tissues by TaqMan usingthe same set of primers and probe as in titration of vector preparations[Zhang, Y., et al., (2001) Mol Ther 3, 697-707].

The performance of vectors base on the new serotypes were evaluated inmurine models of muscle and liver-directed gene transfer and compared tovectors based on the known serotypes AAV1, AAV2 and AAV5. Vectorsexpressing secreted proteins (alpha-antitrypsin (A1AT) and chorionicgonadotropin (CG)) were used to quantitate relative transductionefficiencies between different serotypes through ELISA analysis of sera.The cellular distribution of transduction within the target organ wasevaluated using lacZ expressing vectors and X-gal histochemistry .

The performance of AAV vectors in skeletal muscle was analyzed followingdirect injection into the tibialis anterior muscles. Vectors containedthe same AAV2 based genome with the immediate early gene of CMV or a CMVenhanced β-actin promoter driving expression of the transgene. Previousstudies indicated that immune competent C57BL/6 mice elicit limitedhumoral responses to the human A1AT protein when expressed from AAVvectors [Xiao, W., et al., (1999) J Virol 73, 3994-4003].

In each strain, AAV2/1 vector produced the highest levels of A1AT andAAV2/2 vector the lowest, with AAV2/7 and AAV2/8 vectors showingintermediate levels of expression. Peak levels of CG at 28 daysfollowing injection of nu/nu NCR mice showed the highest levels fromAAV2/7 and the lowest from AAV2/2 with AAV2/8 and AAV2/1 in between.Injection of AAV2/1 and AAV2/7 lacZ vectors yielded gene expression atthe injection sites in all muscle fibers with substantially fewer lacZpositive fibers observed with AAV2/2 and AAV 2/8 vectors. These dataindicate that the efficiency of transduction with AAV2/7 vectors inskeletal muscle is similar to that obtained with AAV2/1, which is themost efficient in skeletal muscle of the previously described serotypes[Xiao, W. (1999), cited above; Chao, H., et al., (2001) Mol Ther 4,217-22; Chao, H., et al., (2000) Mol Ther 2, 619-23].

Similar murine models were used to evaluate liver-directed genetransfer. Identical doses of vector based on genome copies were infusedinto the portal veins of mice that were analyzed subsequently forexpression of the transgene. Each vector contained an AAV2 based genomeusing previously described liver-specific promoters (i.e., albumin orthyroid hormone binding globulin) to drive expression of the transgene.More particularly, CMVCG and TBGCG minigene cassettes were used formuscle and liver-directed gene transfer, respectively. Levels of rhCGwere defined as relative units (RUs×10³). The data were from assayingserum samples collected at day 28, post vector administration (4 animalsper group). As shown in Table 3, the impact of capsid proteins on theefficiency of transduction of A1AT vectors in nu/nu and C57BL/6 mice andCG vectors in C57BL/6 mice was consistent (See Table 6).

TABLE 6 Expression of β-unit of Rhesus Monkey Chorionic Gonadotropin(rhCG) Vector Muscle Liver AAV2/1 4.5 ± 2.1 1.6 ± 1.0 AAV2 0.5 ± 0.1 0.7± 0.3 AAV2/5 ND* 4.8 ± 0.8 AAV2/7 14.2 ± 2.4  8.2 ± 4.3 AAV2/8 4.0 ± 0.776.0 ± 22.8 *Not determined in this experiment.

In all cases, AAV2/8 vectors yielded the highest levels of transgeneexpression that ranged from 16 to 110 greater than what was obtainedwith AAV2/2 vectors; expression from AAV2/5 and AAV2/7 vectors wasintermediate with AAV2/7 higher than AAV2/5. Analysis of X-Gal stainedliver sections of animals that received the corresponding lacZ vectorsshowed a correlation between the number of transduced cells and overalllevels of transgene expression. DNAs extracted from livers of C57BL/6mice who received the A1AT vectors were analyzed for abundance of vectorDNA using real time PCR technology.

The amount of vector DNA found in liver 56 days after injectioncorrelated with the levels of transgene expression (See Table 7). Forthis experiment, a set of probe and primers targeting the SV40 polyAregion of the vector genome was used for TaqMan PCR. Values shown aremeans of three individual animals with standard deviations. The animalswere sacrificed at day 56 to harvest liver tissues for DNA extraction.These studies indicate that AAV8 is the most efficient vector forliver-directed gene transfer due to increased numbers of transducedhepatocytes.

TABLE 7 Real Time PCR Analysis for Abundance of AAV Vectors in nu/nuMouse Liver Following Injection of 1 × 10¹¹ Genome Copies of Vector. AAVvectors/Dose Genome Copies per Cell AAV2/1AlbA1AT  0.6 ± 0.36AAV2AlbA1AT 0.003 ± 0.001 AAV2/5AlbA1AT 0.83 ± 0.64 AAV2/7AlbA1AT 2.2 ±1.7 AAV2/8AlbA1AT 18 ± 11

The serologic data described above suggest that AAV2/8 vector should notbe neutralized in vivo following immunization with the other serotypes.C57BL/6 mice received intraportal injections of AAV2/8 vector expressingcanine factor IX (10¹¹ genome copies) 56 days after they receivedintramuscular injections of A1AT vectors of different serotypes. Highlevels of factor IX expression were obtained 14 days following infusionof AAV2/8 into naïve animals (17±2 μg/ml, n=4) which were notsignificantly different that what was observed in animals immunized withAAV2/1 (31±23 μg/ml, n=4), AAV2/2 (16 μg/ml, n=2), and AAV2/7 (12 μg/ml,n=2). This contrasts to what was observed in AAV2/8 immunized animalsthat were infused with the AAV2/8 factor IX vector in which nodetectable factor IX was observed (<0.1 μg/ml, n=4).

Oligonucleotides to conserved regions of the cap gene did amplifysequences from rhesus monkeys that represented unique AAVs. Identicalcap signature sequences were found in multiple tissues from rhesusmonkeys derived from at least two different colonies. Full-length repand cap open reading frames were isolated and sequenced from singlesources. Only the cap open reading frames of the novel AAVs werenecessary to evaluate their potential as vectors because vectors withthe AAV7 or AAV8 capsids were generated using the ITRs and rep fromAAV2. This also simplified the comparison of different vectors since theactual vector genome is identical between different vector serotypes. Infact, the yields of recombinant vectors generated using this approachdid not differ between serotypes.

Vectors based on AAV7 and AAV8 appear to be immunologically distinct(i.e., they are not neutralized by antibodies generated against otherserotypes). Furthermore, sera from humans do not neutralize transductionby AAV7 and AAV8 vectors, which is a substantial advantage over thehuman derived AAVs currently under development for which a significantproportion of the human population has pre-existing immunity that isneutralizing [Chirmule, N., et al., (1999) Gene Ther 6, 1574-83].

The tropism of each new vector is favorable for in vivo applications.AAV2/7 vectors appear to transduce skeletal muscle as efficiently asAAV2/1, which is the serotype that confers the highest level oftransduction in skeletal muscle of the primate AAVs tested to date[Xiao, W., cited above; Chou (2001), cited above, and Chou (2000), citedabove]. Importantly, AAV2/8 provides a substantial advantage over theother serotypes in terms of efficiency of gene transfer to liver thatuntil now has been relatively disappointing in terms of the numbers ofhepatocytes stably transduced. AAV2/8 consistently achieved a 10 to100-fold improvement in gene transfer efficiency as compared to theother vectors. The basis for the improved efficiency of AAV2/8 isunclear, although it presumably is due to uptake via a differentreceptor that is more active on the basolateral surface of hepatocytes.This improved efficiency will be quite useful in the development ofliver-directed gene transfer where the number of transduced cells iscritical, such as in urea cycle disorders and familialhypercholesterolemia.

Thus, the present invention provides a novel approach for isolating newAAVs based on PCR retrieval of genomic sequences. The amplifiedsequences were easily incorporated into vectors and tested in animals.The lack of pre-existing immunity to AAV7 and the favorable tropism ofthe vectors for muscle indicates that AAV7 is suitable for use as avector in human gene therapy and other in vivo applications. Similarly,the lack of pre-existing immunity to the AAV serotypes of the invention,and their tropisms, renders them useful in delivery of therapeuticmolecules and other useful molecules.

Example 9 Tissue Tropism Studies

In the design of a high throughput functional screening scheme for novelAAV constructs, a non-tissue specific and highly active promoter, CBpromoter (CMV enhanced chicken β actin promoter) was selected to drivean easily detectable and quantifiable reporter gene, human αanti-trypsin gene. Thus only one vector for each new AAV clone needs tobe made for gene transfer studies targeting 3 different tissues, liver,lung and muscle to screen for tissue tropism of a particular AAVconstruct. The following table summarizes data generated from 4 novelAAV vectors in the tissue tropism studies (AAVCBA1AT), from which anovel AAV capsid clone, 44.2, was found to be a very potent genetransfer vehicle in all 3 tissues with a big lead in the lung tissueparticularly. Table 8 reports data obtained (in μg A1AT/mL serum) at day14 of the study.

TABLE 8 Target Tissue Vector Lung Liver Muscle AAV2/1 ND ND 45 ± 11AAV2/5  0.6 ± 0.2 ND ND AAV2/8 ND 84 ± 30 ND AAV2/rh.2 (43.1) 14 ± 7  25± 7.4 35 ± 14 AAV2/rh.10 (44.2) 23 ± 6 53 ± 19 46 ± 11 AAV2/rh.13 (42.2)3.5 ± 2    2 ± 0.8 3.5 ± 1.7 AAV2/rh.21 (42.10) 3.1 ± 2    2 ± 1.4 4.3 ±2  

A couple of other experiments were then performed to confirm thesuperior tropism of AAV 44.2 in lung tissue. First, AAV vector carriedCC10hA1AT minigene for lung specific expression were pseudotyped withcapsids of novel AAVs were given to Immune deficient animals (NCR nude)in equal volume (50 μl each of the original preps without dilution) viaintratracheal injections as provided in the following table. In Table 9,50 μl of each original prep per mouse, NCR Nude, detection limit ≥0.033μg/ml, Day 28

TABLE 9 Relative μg of Gene Total GC μg of A1AT/ml transfer as inA1AT/ml with compared to 50 μl with 1 × 10¹¹ rh.10 (clone Vector vector50 μl vector vector 44.2) 2/1   3 × 10¹² 2.6 ± 0.5 0.09 ± 0.02 2.2 2/25.5 × 10¹¹ <0.03 <0.005 <0.1 2/5 3.6 × 10¹² 0.65 ± 0.16  0.02 ± 0.0040.5 2/7 4.2 × 10¹²   1 ± 0.53 0.02 ± 0.01 0.5 2/8 7.5 × 10¹¹ 0.9 ± 0.70.12 ± 0.09 2.9 2/ch.5 (A.3.1)   9 × 10¹²   1 ± 0.7  0.01 ± 0.008 0.242/rh.8 (43.25) 4.6 × 10¹² 26 ± 21 0.56 ± 0.46 13.7 2/rh.10 (44.2) 2.8 ×10¹² 115 ± 38  4.1 ± 1.4 100 2/rh.13 (42.2)   6 × 10¹² 7.3 ± 0.8 0.12 ±0.01 2.9 2/rh.21 (42.10) 2.4 × 10¹²   9 ± 0.9 0.38 ± 0.04 9.3 2/rh.22(42.11) 2.6 × 10¹²   6 ± 0.4 0.23 ± 0.02 5.6 2/rh.24 (42.13) 1.1 × 10¹¹0.4 ± 0.3 0.4 ± 0.3 1The vectors were also administered to immune competent animals (C57BL/6)in equal genome copies (1×10¹¹ GC) as shown in the Table 10. (1×10¹¹ GCper animal, C57BL/6, day 14, detection limit ≥0.033 μg/ml)

TABLE 10 Relative Gene transfer as μg of A1AT/ml compared to rh.10(clone AAV Vector with 1 × 10¹¹ vector 44.2) 2/1 0.076 ± 0.031 2.6 2/2 0.1 ± 0.09 3.4 2/5 0.0840.033 2.9 2/7 0.33 ± 0.01 11 2/8 1.92 ± 1.3 2.9 2/ch.5 (A.3.1) 0.048 ± 0.004 1.6 2/rh.8 (43.25) 1.7 ± 0.7 58 2/rh.10(44.2) 2.93 ± 1.7  100 2/rh.13 (42.2) 0.45 ± 0.15 15 2/rh.21 (42.10)0.86 ± 0.32 29 2/rh.22 (42.11) 0.38 ± 0.18 13 2/rh.24 (42.13)  0.3 ±0.19 10

The data from both experiments confirmed the superb tropism of clone44.2 in lung-directed gene transfer.

Interestingly, performance of clone 44.2 in liver and muscle directedgene transfer was also outstanding, close to that of the best livertransducer, AAV8 and the best muscle transducer AAV1, suggesting thatthis novel AAV has some intriguing biological significance.

To study serological properties of those novel AAVs, pseudotyped AAVGFPvectors were created for immunization of rabbits and in vitrotransduction of 84-31 cells in the presence and absence of antiseraagainst different capsids. The data are summarized below:

TABLE 11a Cross-NAB assay in 8431 cells and adenovirus (Adv) coinfectionInfection in 8431 cells (coinfected with Adv) with: Serum from rabbit10⁹ GC 10⁹ GC 10⁹ GC 10¹⁰ GC immunized rh.13 rh.21 rh.22 rh.24 with:AAV2/42.2 AAV2/42.10 AAV2/42.11 AAV2/42.13 AAV2/1 1/20 1/20 1/20 No NABAAV2/2 1/640 1/1280 1/5120 No NAB AAV2/5 No NAB 1/40 1/160 No NAB AAV2/71/81920 1/81920 1/40960 1/640 AAV2/8 1/640 1/640 1/320 1/5120 Ch.5AAV2/A3 1/20 1/160 1/640 1/640 rh.8 1/20 1/20 1/20 1/320 AAV2/43.25rh.10 No NAB No NAB No NAB 1/5120 AAV2/44.2 rh.13 1/5120 1/5120 1/5120No NAB AAV2/42.2 rh.21 1/5120 1/10240 1/5120 1/20 AAV2/42.10 rh.221/20480 1/20480 1/40960 No NAB AAV2/42.11 rh.24 No NAB 1/20 1/20 1/5120AAV2/42.13

TABLE 11b Cross-NAB assay in 8431 cells and Adv coinfection Infection in8431 cells (coinfected with Adv) with: Serum 10⁹ GC 10¹⁰ GC 10¹⁰GC 10⁹GC 10⁹ GC from rabbit rh.12 ch.5 rh.8 rh.10 rh.20 immunized with:AAV2/42.1B AAV2/A3 AAV2/43.25 AAV2/44.2 AAV2/42.8.2 AAV2/1 No NAB1/20480 No NAB 1/80 ND AAV2/2 1/20 No NAB No NAB No NAB ND AAV2/5 No NAB1/320 No NAB No NAB ND AAV2/7 1/2560 1/640 1/160 1/81920 ND AAV2/81/10240 1/2560 1/2560 1/81920 ND ch.5 AAV2/A3 1/1280 1/10240 ND 1/51201/320 rh.8 AAV2/43.25 1/1280 ND 1/20400 1/5120 1/2560 rh.10 AAV2/44.21/5120 ND ND 1/5120 1/5120 rh.13 AAV2/42.2 1/20 ND ND No NAB 1/320 rh.21AAV2/42.10 1/20 ND ND 1/40 1/80 rh.22 AAV2/42.11 No NAB ND ND ND No NABrh.24 AAV2/42.13 1/5120 ND ND ND 1/2560

TABLE 12 Titer of rabbit sera Titer after Vector Titer d21 Boosting ch.5AAV2/A3 1/10,240 1/40,960 rh.8 AAV2/43.25 1/20,400 1/163,840 rh.10AAV2/44.2 1/10,240 1/527,680 rh.13 AAV2/42.2 1/5,120 1/20,960 rh.21AAV2/42.10 1/20,400 1/81,920 rh.22 AAV2/42.11 1/40,960 ND rh.24AAV2/42.13 1/5,120 ND

TABLE 13 a Infection in 8431 cells (coinfected with Adv) with GFP 10⁹GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well ch.5AAV2/1 AAV2/2 AAV2/5 AAV2/7 AAV2/8 AAV2/A3 # GFU/field 128 >200 95 56 131 83 >200 65 54 11 1

TABLE 13b Infection in 8431 cells (coinfected with Adv) with GFP 10⁹GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well 10⁹ GC/well 10⁹GC/well rh.8 rh.10 rh.13 rh.21 rh.22 rh.24 rh.12 AAV2/43.25 AAV2/44.2AAV2/42.2 AAV2/42.10 AAV2/42.11 AAV2/42.13 AAV2/42.1B # GFU/field 3 1354 62 10 3 18 2 12 71 60 14 2 20 48 47 16 3 12

Example 10 Mouse Model of Familial Hypercholesterolemia

The following experiment demonstrates that the AAV2/7 construct of theinvention delivers the LDL receptor and express LDL receptor in anamount sufficient to reduce the levels of plasma cholesterol andtriglycerides in animal models of familial hypercholesterolemia.

A. Vector Construction

AAV vectors packaged with AAV7 or AAV8 capsid proteins were constructedusing a pseudotyping strategy [Hildinger M, et al., J Virol 2001;75:6199-6203]. Recombinant AAV genomes with AAV2 inverted terminalrepeats (ITR) were packaged by triple transfection of 293 cells with thecis-plasmid, the adenovirus helper plasmid and a chimeric packagingconstruct, a fusion of the capsids of the novel AAV serotypes with therep gene of AAV2. The chimeric packaging plasmid was constructed aspreviously described [Hildinger et al, cited above]. The recombinantvectors were purified by the standard CsCl₂ sedimentation method. Todetermine the yield TaqMan (Applied Biosystems) analysis was performedusing probes and primers targeting the SV40 poly(A) region of thevectors [Gao G P, et al., Hum Gene Ther. 2000 Oct. 10; 11(15):2079-91].The resulting vectors express the transgene under the control of thehuman thyroid hormone binding globulin gene promoter (TBG).

B. Animals

LDL receptor deficient mice on the C57Bl/6 background were purchasedfrom the Jackson Laboratory (Bar Harbor, Me., USA) and maintained as abreeding colony. Mice were given unrestricted access to water andobtained a high fat Western Diet (high % cholesterol) starting threeweeks prior vector injection. At day −7 as well at day 0, blood wasobtained via retroorbital bleeds and the lipid profile evaluated. Themice were randomly divided into seven groups. The vector was injectedvia an intraportal injection as previously described ([Chen S J et al.,Mol Therapy 2000; 2(3), 256-261]. Briefly, the mice were anaesthetizedwith ketamine and xylazine. A laparotomy was performed and the portalvein exposed. Using a 30g needle the appropriate dose of vector dilutedin 100 ul PBS was directly injected into the portal vein. Pressure wasapplied to the injection site to ensure a stop of the bleeding. The skinwound was closed and draped and the mice carefully monitored for thefollowing day. Weekly bleeds were performed starting at day 14 afterliver directed gene transfer to measure blood lipids. Two animals ofeach group were sacrificed at the time points week 6 and week 12 aftervector injection to examine atherosclerotic plaque size as well asreceptor expression. The remaining mice were sacrificed at week 20 forplaque measurement and determination of transgene expression.

TABLE 14 Vector dose n Group 1 AAV2/7-TBG-hLDLr 1 × 10¹² gc 12 Group 2AAV2/7-TBG-hLDLr 3 × 10¹¹ gc 12 Group 3 AAV2/7-TBG-hLDLr 1 × 10¹¹ gc 12Group 4 AAV2/8-TBG-hLDLr 1 × 10¹² gc 12 Group 5 AAV2/8-TBG-hLDLr 3 ×10¹¹ gc 12 Group 6 AAV2/8-TBG-hLDLr 1 × 10¹¹ gc 12 Group 7AAV2/7-TBG-LacZ 1 × 10¹¹ gc 16

C. Serum Lipoprotein and Liver Function Analysis

Blood samples were obtained from the retroorbital plexus after a 6 hourfasting period. The serum was separated from the plasma bycentrifugation. The amount of plasma lipoproteins and livertransaminases in the serum were detected using an automatized clinicalchemistry analyzer (ACE, Schiapparelli Biosystems, Alpha Wassermann)

D. Detection of Transgene Expression

LDL receptor expression was evaluated by immuno-fluorescence stainingand Western blotting. For Western Blot frozen liver tissue washomogenized with lysis buffer (20 mM Tris, pH7.4, 130 mM NaCl, 1% TritonX 100, proteinase inhibitor (complete, EDTA-free, Roche, Mannheim,Germany). Protein concentration was determined using the Micro BCAProtein Assay Reagent Kit (Pierce, Rockford, Ill.). 40 μg of protein wasresolved on 4-15% Tris-HCl Ready Gels (Biorad, Hercules, Calif.) andtransferred to a nitrocellulose membrane (Invitrogen,). To generateAnti-hLDL receptor antibodies a rabbit was injected intravenously withan AdhLDLr prep (1×10¹³ GC). Four weeks later the rabbit serum wasobtained and used for Western Blot. A 1:100 dilution of the serum wasused as a primary antibody followed by a HRP-conjugated anti-rabbit IgGand ECL chemiluminescent detection (ECL Western Blot Detection Kit,Amersham, Arlington Heights, Ill.).

E. Immunocytochemistry

For determination of LDL receptor expression in frozen liver sectionsimmunohistochemistry analyses were performed. 10 um cryostat sectionswere either fixed in acetone for 5 minutes, or unfixed. Blocking wasobtained via a 1 hour incubation period with 10% of goat serum. Sectionswere then incubated for one hour with the primary antibody at roomtemperature. A rabbit polyclonal antibody anti-human LDL (BiomedicalTechnologies Inc., Stoughton, Mass.) was used diluted accordingly to theinstructions of the manufacturer. The sections were washed with PBS, andincubated with 1:100 diluted fluorescein goat anti-rabbit IgG (Sigma, StLouis, Mo.). Specimens were finally examined under fluorescencemicroscope Nikon Microphot-FXA. In all cases, each incubation wasfollowed by extensive washing with PBS. Negative controls consisted ofpreincubation with PBS, omission of the primary antibody, andsubstitution of the primary antibody by an isotype-matched non-immunecontrol antibody. The three types of controls mentioned above wereperformed for each experiment on the same day.

F. Gene Transfer Efficiency

Liver tissue was obtained after sacrificing the mice at the designatedtime points. The tissue was shock frozen in liquid nitrogen and storedat −80° C. until further processing. DNA was extracted from the livertissue using a QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) according tothe manufacturers protocol. Genome copies of AAV vectors in the livertissue were evaluated using Taqman analysis using probes and primersagainst the SV40 poly(A) tail as described above.

G. Atherosclerotic Plaque Measurement

For the quantification of the atherosclerotic plaques in the mouse aortathe mice were anaesthetized (10% ketamine and xylazine, ip), the chestopened and the arterial system perfused with ice-cold phosphate bufferedsaline through the left ventricle. The aorta was then carefullyharvested, slit down along the ventral midline from the aortic arch downto the femoral arteries and fixed in formalin. The lipid-richatherosclerotic plaques were stained with Sudan IV (Sigma, Germany) andthe aorta pinned out flat on a black wax surface. The image was capturedwith a Sony DXC-960 MD color video camera. The area of the plaque aswell as of the complete aortic surface was determined using Phase 3Imaging Systems (Media Cybernetics).

H. Clearance of I¹²⁵ LDL

Two animals per experimental group were tested. A bolus of I¹²⁵—labeledLDL (generously provided by Dan Rader, U Penn) was infused slowlythrough the tail vein over a period of 30 sec (1,000,000 counts of[I¹²⁵]-LDL diluted in 100 μl sterile PBS/animal) At time points 3 min,30 min, 1.5 hr, 3 hr, 6 hr after injection a blood sample was obtainedvia the retro-orbital plexus. The plasma was separated off from thewhole blood and 10 μl plasma counted in the gamma counter. Finally thefractional catabolic rate was calculated from the lipoprotein clearancedata.

Evaluation of Liver Lipid Accumulation

Oil Red Staining of frozen liver sections was performed to determinelipid accumulation. The frozen liver sections were briefly rinsed indistilled water followed by a 2 minute incubation in absolute propyleneglycol. The sections were then stained in oil red solution (0.5% inpropylene glycol) for 16 hours followed by counterstaining with Mayer'shematoxylin solution for 30 seconds and mounting in warmed glycerinjelly solution.

For quantification of the liver cholesterol and triglyceride contentliver sections were homogenized and incubated in chloroform/methanol(2:1) overnight. After adding of 0.05% H₂SO₄ and centrifugation for 10minutes, the lower layer of each sample was collected, divided in twoaliquots and dried under nitrogen. For the cholesterol measurement thedried lipids of the first aliquot were dissolved in 1% Triton X-100 inchloroform. Once dissolved, the solution was dried under nitrogen. Afterdissolving the lipids in ddH₂0 and incubation for 30 minutes at 37° C.the total cholesterol concentration was measured using a TotalCholesterol Kit (Wako Diagnostics). For the second aliquot the driedlipids were dissolved in alcoholic KOH and incubated at 60° C. for 30minutes. Then 1M MgCl2 was added, followed by incubation on ice for 10minutes and centrifugation at 14,000 rpm for 30 minutes. The supernatantwas finally evaluated for triglycerides (Wako Diagnostics).

All of the vectors pseudotyped in an AAV2/8 or AAV2/7 capsid loweredtotal cholesterol, LDL and triglycerides as compared to the control.These test vectors also corrected phenotype of hypercholesterolemia in adose-dependent manner. A reduction in plaque area for the AAV2/8 andAAV2/7 mice was observed in treated mice at the first test (2 months),and the effect was observed to persist over the length of the experiment(6 months).

Example 10 Functional Factor IX Expression and Correction of Hemophilia

A. Knock-Out Mice

Functional canine factor IX (FIX) expression was assessed in hemophiliaB mice. Vectors with capsids of AAV1, AAV2, AAV5, AAV7 or AAV8 wereconstructed to deliver AAV2 5′ ITR—liver-specific promoter [LSP]-canineFIX—woodchuck hepatitis post-regulatory element (WPRE)—AAV2 3′ ITR. Thevectors were constructed as described in Wang et al, 2000, MolecularTherapy 2: 154-158), using the appropriate capsids.

Knock-out mice were generated as described in Wang et al, 1997. Proc.Natl. Acad. Sci. USA 94: 11563-11566. This model closely mimic thephenotypes of hemophilia Bin human.

Vectors of different serotypes (AAV1, AAV2, AAV5, AAV7 and AAV8) weredelivered as a single intraportal injection into the liver of adulthemophiliac C57Bl/6 mice in a dose of 1×10¹¹ GC/mouse for the fivedifferent serotypes and one group received an AAV8 vector at a lowerdose, 1×10¹⁰ GC/mouse. Control group was injected with 1×10¹¹ GC ofAAV2/8 TBG LacZ3. Each group contains 5-10 male and female mice. Micewere bled bi-weekly after vector administration.

1. ELISA

The canine FIX concentration in the mouse plasma was determined by anELISA assay specific for canine factor IX, performed essentially asdescribed by Axelrod et al, 1990, Proc.Natl.Acad.Sci.USA, 87:5173-5177with modifications. Sheep anti-canine factor IX (Enzyme ResearchLaboratories) was used as primary antibody and rabbit anti-canine factorIX ((Enzyme Research Laboratories) was used as secondary antibody.Beginning at two weeks following injection, increased plasma levels ofcFIX were detected for all test vectors. The increased levels weresustained at therapeutic levels throughout the length of the experiment,i.e., to 12 weeks. Therapeutic levels are considered to be 5% of normallevels, i.e., at about 250 ng/mL.

The highest levels of expression were observed for the AAV2/8 (at 10¹¹)and AAV2/7 constructs, with sustained superphysiology levels cFIX levels(ten-fold higher than the normal level). Expression levels for AAV2/8(10¹¹) were approximately 10 fold higher than those observed for AAV2/2and AAV2/8 (10¹⁰). The lowest expression levels, although still abovethe therapeutic range, were observed for AAV2/5.

2. In Vitro Activated Partial Thromboplastin Time (aPTT) Assay

Functional factor IX activity in plasma of the FIX knock-out mice wasdetermined by an in vitro activated partial thromboplastin time (aPTT)assay—Mouse blood samples were collected from the retro-orbital plexusinto 1/10 volume of citrate buffer. The aPTT assay was performed asdescribed by Wang et al, 1997, Proc. Natl. Acad. Sci. USA 94:11563-11566.

Clotting times by aPTT on plasma samples of all vector injected micewere within the normal range (approximately 60 sec) when measured at twoweeks post-injection, and sustained clotting times in the normal orshorter than normal range throughout the study period (12 weeks).

Lowest sustained clotting times were observed in the animals receivingAAV2/8 (10¹¹) and AAV2/7. By week 12, AAV2/2 also induced clotting timessimilar to those for AAV2/8 and AAV2/7. However, this lowered clottingtime was not observed for AAV2/2 until week 12, whereas lowered clottingtimes (in the 25-40 sec range) were observed for AAV2/8 and AAV2/7beginning at week two.

Immuno-histochemistry staining on the liver tissues harvested from someof the treated mice is currently being performed. About 70-80% ofhepatocytes are stained positive for canine FIX in the mouse injectedwith AAV2/8.cFIX vector.

B. Hemophilia B Dogs

Dogs that have a point mutation in the catalytic domain of the F.IXgene, which, based on modeling studies, appears to render the proteinunstable, suffer from hemophilia B [Evans et al, 1989, Proc. Natl. Acad.Sci. USA, 86:10095-10099). A colony of such dogs has been maintained formore than two decades at the University of North Carolina, Chapel Hill.The homeostatic parameters of these dogs are well described and includethe absence of plasma F.IX antigen, whole blood clotting times in excessof 60 minutes, whereas normal dogs are 6-8 minutes, and prolongedactivated partial thromboplastin time of 50-80 seconds, whereas normaldogs are 13-28 seconds. These dogs experience recurrent spontaneoushemorrhages. Typically, significant bleeding episodes are successfullymanaged by the single intravenous infusion of 10 ml/kg of normal canineplasma; occasionally, repeat infusions are required to control bleeding.

Four dogs are injected intraportally with AAV.cFIX according to theschedule below. A first dog receives a single injection with AAV2/2.cFIXat a dose of 3.7×10¹¹ genome copies (GC)/kg. A second dog receives afirst injection of AAV2/2.cFIX (2.8×10¹¹ GC/kg), followed by a secondinjection with AAV2/7.cFIX (2.3×10¹³ GC/kg) at day 1180. A third dogreceives a single injection with AAV2/2.cFIX at a dose of 4.6×10¹²GC/kg. The fourth dog receives an injection with AAV2/2.cFIX (2.8×10¹²GC/kg) and an injection at day 995 with AAV2/7.cFIX (5×10¹² GC/kg).

The abdomen of hemophilia dogs are aseptically and surgically openedunder general anesthesia and a single infusion of vector is administeredinto the portal vein. The animals are protected from hemorrhage in thepen-operative period by intravenous administration of normal canineplasma. The dog is sedated, intubated to induce general anesthesia, andthe abdomen shaved and prepped. After the abdomen is opened, the spleenis moved into the operative field. The splenic vein is located and asuture is loosely placed proximal to a small distal incision in thevein. A needle is rapidly inserted into the vein, then the sutureloosened and a 5 F cannula is threaded to an intravenous location nearthe portal vein threaded to an intravenous location near the portal veinbifurcation. After hemostasis is secured and the catheter ballooninflated, approximately 5.0 ml of vector diluted in PBS is infused intothe portal vein over a 5 minute interval. The vector infusion isfollowed by a 5.0 ml infusion of saline. The balloon is then deflated,the callula removed and venous hemostasis is secured. The spleen is thenreplaced, bleeding vessels are cauterized and the operative wound isclosed. The animal is extubated having tolerated the surgical procedurewell. Blood samples are analyzed as described. [Wang et al, 2000,Molecular Therapy 2: 154-158]

Results showing correction or partial correction are anticipated forAAV2/7.

All publications cited in this specification including priorityapplications, U.S. patent application Ser. No. 15/584,674, U.S. patentapplication Ser. No. 14/956,934, U.S. patent application Ser. No.13/633,971, U.S. patent application Ser. No. 12/962,793, U.S. patentapplication Ser. No. 10/291,583, and U.S. provisional patent applicationNos. 60/386,675, 60/377,066, 60/341,117, and 60/350,607, areincorporated herein by reference. While the invention has been describedwith reference to particularly preferred embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the claims.

1. A recombinant nucleic acid molecule: (a) encoding an AAVrh.20 vp1capsid protein having a sequence comprising amino acids 1 to 738 of SEQID NO: 85; or (b) comprising nucleotides 844 to 3057 of SEQ ID NO: 27,or a nucleotide sequence at least 99% identical to nucleotides 844 to3057 of SEQ ID NO: 27, wherein the recombinant nucleic acid moleculedoes not contain an AAV inverted terminal repeat.
 2. The recombinantnucleic acid molecule according to claim 1, which further comprises afunctional rep gene.
 3. The recombinant nucleic acid molecule accordingto claim 1, wherein said recombinant nucleic acid molecule comprises asequence selected from: vp1, nt 844 to 3057; vp2, nt 1255 to 3057; orvp3, nt 1453 to 3057 of SEQ ID NO:
 27. 4. The recombinant nucleic acidmolecule according to claim 1, wherein said recombinant nucleic acidmolecule is a plasmid.
 5. A host cell transfected with the recombinantnucleic acid molecule according to claim
 1. 6. The host cell accordingto claim 5, which further comprises a functional rep gene, a minigenecomprising AAV inverted terminal repeats (ITRs) and a transgene, andsufficient helper functions to permit packaging of the minigene into theAAV capsid.
 7. The host cell according to claim 6, which furthercomprises a nucleic acid sequence (a) encoding an AAVrh.20 vp2 capsidprotein having a sequence comprising amino acids 138 to 738 of SEQ IDNO: 85; or (b) comprising nucleotides 1255 to 3057 of SEQ ID NO: 27, ora nucleotide sequence at least 99% identical to nucleotides 1255 to 3057of SEQ ID NO:
 27. 8. The host cell according to claim 6, which furthercomprises a nucleic acid sequence (a) encoding an AAVrh.20 vp3 capsidprotein having a sequence comprising amino acids 204 to 738 of SEQ IDNO: 85; or (b) comprising nucleotides 1453 to 3057 of SEQ ID NO: 27, ora nucleotide sequence at least 99% identical to nucleotides 1453 to 3057of SEQ ID NO:
 27. 9. A recombinant AAV comprising an AAV8 capsidproduced by the host cell of claim
 6. 10. A recombinant AAV comprisingan AAV8 capsid protein encoded by the recombinant nucleic acid moleculeaccording to claim
 1. 11. A composition comprising the recombinant AAVaccording to claim 10 and a pharmaceutically acceptable carrier.
 12. Arecombinant nucleic acid molecule: (a) encoding an AAVrh.20 vp2 capsidprotein having a sequence comprising amino acids 138 to 738 of SEQ IDNO: 85; or (b) comprising nucleotides 1255 to 3057 of SEQ ID NO: 27, ora nucleotide sequence at least 99% identical to nucleotides 1255 to 3057of SEQ ID NO: 27, wherein the recombinant nucleic acid molecule does notcontain an AAV inverted terminal repeat.
 13. The recombinant nucleicacid molecule according to claim 12, which further comprises afunctional rep gene.
 14. The recombinant nucleic acid molecule accordingto claim 12, wherein said recombinant nucleic acid molecule is aplasmid.
 15. A host cell transfected with the recombinant nucleic acidmolecule according to claim
 12. 16. The host cell according to claim 15,which further comprises a functional rep gene, a minigene comprising AAVinverted terminal repeats (ITRs) and a transgene, and sufficient helperfunctions to permit packaging of the minigene into the AAV capsid.
 17. Arecombinant AAV comprising an AAV8 capsid produced by the host cell ofclaim
 16. 18. A recombinant AAV comprising an AAV8 capsid proteinencoded by the recombinant nucleic acid molecule according to claim 12.19. A composition comprising the recombinant AAV according to claim 18and a pharmaceutically acceptable carrier.
 20. A recombinant nucleicacid molecule: (a) encoding an AAVrh.20 vp3 capsid protein having asequence comprising amino acids 204 to 738 of SEQ ID NO: 85; or (b)comprising nucleotides 1453 to 3057 of SEQ ID NO: 27, or a nucleotidesequence at least 99% identical to nucleotides 1453 to 3057 of SEQ IDNO: 27, wherein the recombinant nucleic acid molecule does not containan AAV inverted terminal repeat.
 21. The recombinant nucleic acidmolecule according to claim 20, which further comprises a functional repgene.
 22. The recombinant nucleic acid molecule according to claim 20,wherein said recombinant nucleic acid molecule is a plasmid.
 23. A hostcell transfected with the recombinant nucleic acid molecule according toclaim
 20. 24. The host cell according to claim 23, which furthercomprises a functional rep gene, a minigene comprising AAV invertedterminal repeats (ITRs) and a transgene, and sufficient helper functionsto permit packaging of the minigene into the AAV capsid.
 25. Arecombinant AAV comprising an AAV8 capsid produced by the host cell ofclaim
 24. 26. A recombinant AAV comprising an AAV8 capsid proteinencoded by the recombinant nucleic acid molecule according to claim 20.27. A composition comprising the recombinant AAV according to claim 26and a pharmaceutically acceptable carrier.
 28. An AAVrh.20 vectorcomprising an AAVrh.20 capsid, said capsid having packaged therein anucleic acid molecule comprising AAV inverted terminal repeat sequencesand a gene encoding a product, which gene is operably linked toregulatory sequences which direct expression of the gene product,wherein said AAVrh.20 capsid is produced from expression in an AAVpackaging cell comprising a AAVrh.20 vp1 nucleotide sequence which is atleast about 90% identical to nucleotides 844 to 3057 of SEQ ID NO: 27and which encodes amino acids 1 to 738 of SEQ ID NO: 85.